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Increased Cytotoxicity of 3-Morpholinosydnonimine to HepG2 Cells in the Presence of Superoxide Dismutase

1995; Elsevier BV; Volume: 270; Issue: 36 Linguagem: Inglês

10.1074/jbc.270.36.20922

1083-351X

D Gergèl, Vladimı́r Mišı́k, Karol Ondriaš, Arthur I. Cederbaum,

Free Radicals and Antioxidants

3-Morpholinosydnonimine (SIN-1) is widely used to generate nitric oxide (NOx˙) and superoxide radical (O). The effect of SOD on the toxicity of SIN-1 is complex, depending on what is the ultimate species responsible for toxicity. SIN-1 (<1 mM) was only slightly toxic to HepG2 cells. Copper, zinc superoxide dismutase (Cu,Zn-SOD) or manganese superoxide dismutase (Mn-SOD) increased the toxicity of SIN-1. Catalase abolished, while sodium azide potentiated, this toxicity, suggesting a key role for H2O2 in the overall mechanism. Depletion of GSH from the HepG2 cells also potentiated the toxicity of SIN-1 plus SOD. Although Me2SO, sodium formate, and mannitol had no protective effect, iron chelators, thiourea and urate protected the cells against the SIN-1 plus Cu,Zn-SOD-mediated cytotoxicity. The cytotoxic effect of Cu,Zn-SOD but not Mn-SOD, showed a biphasic dose response being most pronounced at lower concentrations (10-100 units/ml). In the presence of SIN-1, Mn-SOD increased accumulation of H2O2 in a concentration-dependent manner. In contrast, Cu,Zn-SOD increased H2O2 accumulation from SIN-1 at low but not high concentrations of the enzyme, suggesting that high concentrations of the Cu,Zn-SOD interacted with the H2O2. EPR spin trapping studies demonstrated the formation of hydroxyl radical from the decomposition of H2O2 by high concentrations of the Cu,Zn-SOD. The cytotoxic effect of the NO donors SNAP and DEA/NO was only slightly enhanced by SOD; catalase had no effect. Thus, the oxidants responsible for the toxicity of SIN-1 and SNAP or DEA/NO to HepG2 cells under these conditions are different, with H2O2 derived from O dismutation playing a major role with SIN-1. These results suggest that the potentiation of SIN-1 toxicity by SOD is due to enhanced production of H2O2, followed by site-specific damage of critical cellular sites by a transition metal-catalyzed reaction. These results also emphasize that the role of SOD as a protectant against oxidant damage is complex and dependent, in part, on the subsequent fate and reactivity of the generated H2O2. 3-Morpholinosydnonimine (SIN-1) is widely used to generate nitric oxide (NOx˙) and superoxide radical (O). The effect of SOD on the toxicity of SIN-1 is complex, depending on what is the ultimate species responsible for toxicity. SIN-1 (<1 mM) was only slightly toxic to HepG2 cells. Copper, zinc superoxide dismutase (Cu,Zn-SOD) or manganese superoxide dismutase (Mn-SOD) increased the toxicity of SIN-1. Catalase abolished, while sodium azide potentiated, this toxicity, suggesting a key role for H2O2 in the overall mechanism. Depletion of GSH from the HepG2 cells also potentiated the toxicity of SIN-1 plus SOD. Although Me2SO, sodium formate, and mannitol had no protective effect, iron chelators, thiourea and urate protected the cells against the SIN-1 plus Cu,Zn-SOD-mediated cytotoxicity. The cytotoxic effect of Cu,Zn-SOD but not Mn-SOD, showed a biphasic dose response being most pronounced at lower concentrations (10-100 units/ml). In the presence of SIN-1, Mn-SOD increased accumulation of H2O2 in a concentration-dependent manner. In contrast, Cu,Zn-SOD increased H2O2 accumulation from SIN-1 at low but not high concentrations of the enzyme, suggesting that high concentrations of the Cu,Zn-SOD interacted with the H2O2. EPR spin trapping studies demonstrated the formation of hydroxyl radical from the decomposition of H2O2 by high concentrations of the Cu,Zn-SOD. The cytotoxic effect of the NO donors SNAP and DEA/NO was only slightly enhanced by SOD; catalase had no effect. Thus, the oxidants responsible for the toxicity of SIN-1 and SNAP or DEA/NO to HepG2 cells under these conditions are different, with H2O2 derived from O dismutation playing a major role with SIN-1. These results suggest that the potentiation of SIN-1 toxicity by SOD is due to enhanced production of H2O2, followed by site-specific damage of critical cellular sites by a transition metal-catalyzed reaction. These results also emphasize that the role of SOD as a protectant against oxidant damage is complex and dependent, in part, on the subsequent fate and reactivity of the generated H2O2. INTRODUCTIONNitric oxide (NO) 1The abbreviations used are: NO˙nitric oxideNOxnitrogen oxidative metabolitesSODsuperoxide dismutaseCu,Zn-SODcopper, zinc superoxide dismutaseMn-SODmanganese superoxide dismutaseSIN-13-morpholinosydnonimineSNAPS-nitroso-N-acetylpenicillamineDEA/NOdiethylamine/nitric oxide complexOsuperoxide anionONOO-peroxynitrite anionOHhydroxyl radicalDTPAdiethylenetriamine pentaacetic acidMEMminimum essential mediumBSOL-buthionine (S,R)-sulfoximineDMPO5,5-Dimethylpyrroline-N-oxide. and other reactive nitrogen oxidative metabolites (NOx) are produced by a variety of mammalian cells. These agents may be important for host defense, but under certain conditions, NO and NOx may cause tissue damage by still to be clarified mechanisms. Both NO and superoxide anion radical (O) are known to be generated by macrophages, neutrophils, and endothelial cells(1Rosen G.M. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7268-7273Crossref Scopus (354) Google Scholar, 2Marletta M.A. Trends Biochem. Sci. 1989; 14: 488-492Abstract Full Text PDF PubMed Scopus (373) Google Scholar, 3Moncada J. Palmer R.M.J. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142PubMed Google Scholar). The production of these two radicals under physiological conditions can lead to the formation of peroxynitrite (ONOO-) and other reactive species that are cytotoxic(4Beckman J.S. Beckman T.W. Chen J. Marshall P.A. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 84: 1620-1624Crossref Scopus (6665) Google Scholar, 5Ischiropoulos H. Zhu L. Chen J. Tsai M. Martin J.C. Smith C.D. Beckman J.S. Arch. Biochem. 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Freeman B.A. J. Biol. Chem. 1991; 266: 4244-4250Abstract Full Text PDF PubMed Google Scholar), produce nitration of tyrosine residues in proteins(5Ischiropoulos H. Zhu L. Chen J. Tsai M. Martin J.C. Smith C.D. Beckman J.S. Arch. Biochem. Biophys. 1992; 298: 431-437Crossref PubMed Scopus (1420) Google Scholar), or react with sugars (4Beckman J.S. Beckman T.W. Chen J. Marshall P.A. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 84: 1620-1624Crossref Scopus (6665) Google Scholar) and DNA(12King P.A. Anderson V.E. Edwards J. Gustafson G. Plumb R. Luggs J. J. Am. Chem. Soc. 1992; 114: 5430-5432Crossref Scopus (244) Google Scholar). Superoxide dismutase (SOD) present in the cytoplasm and mitochondria within cells and in the extracellular space dismutates two O into H2O2 and O2(13McCord J.M. Fridovich I. J. Biol. Chem. 1969; 244: 6049-6055Abstract Full Text PDF PubMed Google Scholar). The protective effect of SOD against oxygen-derived free radicals in vivo, in intact cells and in vitro studies is well documented(14McCord J.M. Fridovich I. Free Radical Biol. Med. 1988; 5: 363-369Crossref PubMed Scopus (279) Google Scholar, 15Michiels C. Raes M. Taussaint O. Remacle J. Free Radical Biol. Med. 1994; 17: 235-248Crossref PubMed Scopus (1023) Google Scholar). However, in some studies a bell-shaped dose-response curve for the protective effect of SOD was observed. At low concentrations, SOD was usually protective, but at very high concentrations, its protective effect decreased or was reversed such that SOD potentiated toxicity(16Omar B.A. Gad N.M. Jordan M.C. Striplin S.P. Russell W.J. Downey J.M. McCord J.M. Free Radical Biol. Med. 1990; 9: 465-471Crossref PubMed Scopus (131) Google Scholar, 17Barnier M. Manning A.S. Hearse D.J. Am. J. Physiol. 1989; 256: H1344-H1352PubMed Google Scholar, 18Nelson S.K. Bose S.K. McCord J.M. Free Radical Biol. Med. 1994; 16: 195-200Crossref PubMed Scopus (128) Google Scholar).SIN-1 (3-morpholinosydnonimine), the active metabolite of the vasodilatatory drug molsidomine, is frequently used as a model compound for a continuous release of O, NO and/or NOx, and other potent oxidants such as ONOO- and hydroxyl radical (OH)(16Omar B.A. Gad N.M. Jordan M.C. Striplin S.P. Russell W.J. Downey J.M. McCord J.M. Free Radical Biol. Med. 1990; 9: 465-471Crossref PubMed Scopus (131) Google Scholar, 19Feelisch M. Ostrowski J. Noack E. J. Cardiovasc. Pharmacol. 1989; 14: 313-322Google Scholar, 20Augusto O. Gatti R.M. Radi R. Arch. Biochem. Biophys. 1994; 310: 118-125Crossref PubMed Scopus (167) Google Scholar). These SIN-1-derived strong oxidants degrade deoxoyribose(6Hogg N. Darley-Usmar V.M. Wilson M.T. Moncada S. Biochem. J. 1992; 28: 419-424Crossref Scopus (619) Google Scholar), oxidize low density lipoproteins(21Darley-Usmar V.M. Hogg N. O'Leary V.J. Moncada S. Free Rad. Res. Commun. 1992; 14: 19-20Google Scholar), mediate loss of microsomal α-tocopherol(22deGroot H. Hegi U. Sies H. FEBS Lett. 1992; 315: 139-142Crossref Scopus (85) Google Scholar), damage surfactant protein A(23Haddad I.Y. Crow J.P. Hu P. Ye Y. Beckman T. Matalou S. Am. J. Physiol. 1994; 264: L242-L249Google Scholar), and inhibit glyceraldehyde-3-phosphate dehydrogenase (24Dimmeler S. Lottspeich F. Brune B. J. Biol. Chem. 1992; 267: 16771-16774Abstract Full Text PDF PubMed Google Scholar) and hepatic gluoneogenesis(25Horton R.A. Ceppi E.D. Knowles R.G. Titheradge M.A. Biochem. J. 1994; 299: 735-739Crossref PubMed Scopus (76) Google Scholar). Cytotoxic effects of SIN-1 have been demonstrated in a variety of cell lines(26Lafon-Cazal M. Culcasi M. Gaven F. Pietri S. Bockaert J. Neuropharmacology. 1993; 32: 1259-1266Crossref PubMed Scopus (177) Google Scholar, 27Kröncke K.-D. Brenner H.-H. Rodriguez M.-L. Etzkorn K. Noack E.A. Kolb H. Kolb-Backhofen V. Biochim. Biophys. Acta. 1993; 1182: 221-229Crossref PubMed Scopus (103) Google Scholar, 28Ioannidis T. DeGroot H. Biochem. J. 1993; 296: 341-345Crossref PubMed Scopus (129) Google Scholar). For example, the LD50 for SIN-1 cytotoxicity against Escherichia coli was 0.5 mM(29Brunelli L. Crow J.P. Beckman J.S. Arch. Biochem. Biophys. 1995; 316: 327-334Crossref PubMed Scopus (262) Google Scholar), and 1 mM SIN-1 was very toxic to neurons(30Lipton S.A. Choi Y.-B. Pan Z.-H. Lei S.Z. Chen H.-S.V. Sucher N.J. Loscalso J. Singel D.J. Stamler J.S. Nature. 1993; 364: 626-632Crossref PubMed Scopus (2287) Google Scholar). SOD had a protective effect against SIN-1 toxicity to cultured neurons (30Lipton S.A. Choi Y.-B. Pan Z.-H. Lei S.Z. Chen H.-S.V. Sucher N.J. Loscalso J. Singel D.J. Stamler J.S. Nature. 1993; 364: 626-632Crossref PubMed Scopus (2287) Google Scholar) and to E. coli(29Brunelli L. Crow J.P. Beckman J.S. Arch. Biochem. Biophys. 1995; 316: 327-334Crossref PubMed Scopus (262) Google Scholar), but had no protective effect with rat hepatoma cells(28Ioannidis T. DeGroot H. Biochem. J. 1993; 296: 341-345Crossref PubMed Scopus (129) Google Scholar), and SOD actually increased SIN-1 toxicity to Leishmania major(31Assreuy T. Cunha F.Q. Epperlein M. Noronha-Dutra A. O'Donnell C.A. Liew F.Y. Moncada S. Eur. J. Immunol. 1994; 24: 672-676Crossref PubMed Scopus (238) Google Scholar). The effect of SOD on the toxicity of SIN-1 is complex, e.g. if ONOO3 is the cause of toxicity, SOD will be protective since dismutation of O will prevent formation of ONOO3. However, if NO itself is the toxic agent, SOD can potentiate NO toxicity by preventing its reaction with O, thereby elevating steady state concentrations of NO. The role of H2O2, the product of O dismutation by SOD, has generally not been considered as important as nitrogenous metabolites in SIN-1 toxicity. It was of interest that the Cu,Zn-SOD did not provide more than 50% protection against SIN-1 toxicity to E. coli even when added in excess(29Brunelli L. Crow J.P. Beckman J.S. Arch. Biochem. Biophys. 1995; 316: 327-334Crossref PubMed Scopus (262) Google Scholar), perhaps due to the toxicity of H2O2 produced from O dismutation. In the current report, the effect of SOD on SIN-1 cytotoxicity in a human hepatoma liver cell line (HepG2) was determined. It was observed that Cu,Zn-SOD and Mn-SOD enhanced SIN-1-mediated cytotoxicity to HepG2 cells, and that this potentiation was due to an increase in H2O2 formation caused by SOD-catalyzed dismutation of the O released from SIN-1. The toxicity of SIN-1 under these reaction conditions and concentrations appeared to be independent of NO or NOx.EXPERIMENTAL PROCEDURESMaterialsSIN-1 and SNAP were from Biomol (Plymouth Meeting, PA). DEA/NO was from Research Biochemical (Natic, MA). Cu,Zn-superoxide dismutase from bovine erythrocytes and catalase were obtained from Boehringer Mannheim. Mn-superoxide dismutase from E. coli, benzoate, Me2SO, formate, thiourea, BSO, deferoxamine mesylate, 2,2′-dipyridyl, H2O2, EDTA, and DTPA were from Sigma. Sodium azide was from Aldrich. 5,5-Dimethylpyrroline-N-oxide (DMPO) was from Sigma and was purified before use by charcoal treatment to remove paramagnetic impurities.Cell CulturesThe human hepatoma cell line HepG2 (American Type Culture Collection, HB8065) was used for these studies. Cells were cultured in minimum essential medium (MEM, Life Technologies, Inc.) supplemented with 1% PSN antibiotic mixture (Life Technologies, Inc.), and, 10% fetal calf serum (Sigma) in 25-cm2 plastic flasks (Corning) at 37°C in a humidified atmosphere of 95% air and 5% CO2. For cytotoxicity experiments, cells were plated at 4 × 104 cells/well/0.5 ml of tissue culture medium on 24-well tissue culture plates. After 2 h of incubation and attachment, SIN-1, SOD, as well as other compounds were added and the cells were incubated for the indicated time periods at 37°C. Stock solutions of all compounds were prepared in MEM medium immediately before use. The preparation of solutions and incubation with the HepG2 cells were carried out under protection from light. Cell viability (cytotoxicity) was determined using the Cell Titer 96 non-radioactive cell proliferation/cytotoxicity assay kit (Promega), which is based on the cellular conversion of a tetrazolium salt into a formazan product that can be detected spectrophotometrically(32Mosmann T. J. Immunol. Methods. 1983; 65: 55-58Crossref PubMed Scopus (45530) Google Scholar). GSH levels were determined using the GSH-400 kit assay (Cayman Chemical, Ann Arbor, MI).Determination of H2O2H2O2 generation at different concentrations of SIN-1, in the absence or presence of SOD, was determined by two techniques after a 24-h incubation in tissue culture medium at 37°C in 95% air and 5% CO2 in the absence of cells. In the first method, the H2O2 content was measured by formation of formaldehyde from the oxidation of methanol by the catalase-H2O2 compound I complex(33Hildebrandt A.G. Roots I. Tjoe M. Heinemeyer G. Methods Enzymol. 1978; 52: 342-350Crossref PubMed Scopus (192) Google Scholar). Incubations contained 100 mM methanol, 1200 units/ml catalase, SIN-1, and tissue culture medium. Reactions were terminated after a 1-h incubation at 37°C by the addition of 20% trichloroacetic acid. The generation of formaldehyde was determined by the Nash reaction(34Nash T. Biochem. J. 1953; 55: 416-421Crossref PubMed Scopus (4090) Google Scholar), and calculation of H2O2 concentration was carried out as described previously(35Kukie E. Cederbaum A.I. Arch. Biochem. Biophys. 1989; 275: 540-550Crossref PubMed Scopus (36) Google Scholar). In the second method, the formation of H2O2 was determined by the horseradish peroxidase-catalyzed reaction of H2O2 with 4-amino-antipyrine plus 3,5-dichloro-2-hydroxybenzenesulfonic acid. After a 1-h incubation of SIN-1 with the above compounds in tissue culture medium at 37°C, the resulting product was measured spectrophotometrically at 546 nm (28Ioannidis T. DeGroot H. Biochem. J. 1993; 296: 341-345Crossref PubMed Scopus (129) Google Scholar, 36Fossati P. Prencipe L. Berti G. Clin. Chem. 1980; 26: 227-231Crossref PubMed Scopus (814) Google Scholar).EPR MeasurementsThe production of OH from the SOD-catalyzed decomposition of H2O2 was determined by spin trapping with DMPO. MEM containing 100 or 1000 units/ml Cu,Zn-SOD or Mn-SOD and 100 mM DMPO was incubated with 80 μM H2O2. The samples were filled in a Varian flat cell and measured within 1 min after addition of H2O2, using a Varian EPR 200 spectrometer operating in the X-band mode. Instrument parameters are indicated in the legends to figures.In separate experiments evaluating iron-catalyzed decomposition of H2O2, 1 mM SIN-1 was co-incubated with either Cu,Zn-SOD (100 or 1000 units/ml) or Mn-SOD (100 or 1000 units/ml) for 24 h at 37°C in the absence of cells. At the end of the incubation period, 100 mM DMPO and 600 mM Me2SO were added and decomposition of H2O2 was initiated by the addition of 40 μM FeSO4. In the presence of Me2SO, OH radicals are converted to CH3 radicals, which were spin-trapped with DMPO (DMPO/CH3 adducts), thus providing a characteristic OH radical fingerprint(37Britigan B.E. Cohen M.S. Rosen G.M. J. Leukocyte Biol. 1987; 41: 349-362Crossref PubMed Scopus (85) Google Scholar). The samples were transferred to a EPR flat cell and measured immediately after the addition of the last compound.RESULTSCytotoxicity of SIN-1 to HepG2 CellsThe cytotoxic effect of SIN-1 in the absence or presence of Cu,Zn-SOD to the human hepatoma HepG2 cell line was studied at different SIN-1 and Cu,Zn-SOD concentrations over a 24-h time period (Fig. 1). SIN-1 alone at concentrations of 0.1 or 0.316 mM was not toxic over this time course; some toxicity was observed at 1 mM SIN-1. At the very high concentration of 3 mM, SIN-1 toxicity was pronounced and increased during incubation. The addition of Cu,Zn-SOD potentiated the cytotoxic effect of SIN-1; this is most notable at SIN-1 concentrations of 0.316 and 1 mM (Fig. 1, B and C). Pronounced cytotoxicity was found in the presence of Cu,Zn-SOD plus SIN-1 at concentrations where SIN-1 alone had no effect (0.316 mM) or only a minor effect (1 mM). The potentiation of the cytotoxic effect of SIN-1 by Cu,Zn-SOD increased during incubation. A surprising observation was the finding that in the presence of 0.316 mM SIN-1, the potentiating cytotoxic effect of Cu,Zn-SOD was pronounced at 10 and 100 units of SOD per ml, but decreased as the units of added Cu,Zn-SOD were elevated to 1000 units/ml. In other experiments, 0.316 or 1 mM SIN-1 in the absence and presence of 100 units of Cu,Zn-SOD was preincubated in culture medium in the absence of HepG2 cells for 3 h. The HepG2 cells were added, and cell viability was compared to that of a system in which SIN-1, SOD, and cells were added together at the same time. At all time points tested (2-18 h), the medium "conditioned" by preincubating SIN-1 and Cu,Zn-SOD was considerably more toxic, which suggests accumulation of a stable toxic metabolite produced from the incubation of SIN-1 in the presence of SOD.A more detailed study of the concentration dependence of the cytotoxic effect of Cu,Zn-SOD in the presence of 0.316 mM SIN-1 is shown in Fig. 2. The potentiation of SIN-1 cytotoxicity by Cu,Zn-SOD was biphasic; toxicity increased in a concentration-dependent manner up to 100 units of Cu,Zn-SOD/ml but decreased at higher concentrations (Fig. 2A). Boiled Cu,Zn-SOD did not potentiate the cytotoxicity of SIN-1, and Cu,Zn-SOD in the absence of SIN-1 had no cytotoxic effect (Fig. 2A). Another type of superoxide dismutase, Mn-SOD, also potentiated the cytotoxicity of SIN-1, but in contrast to the Cu,Zn-SOD, the potentiation of SIN-1 cytotoxicity by the Mn-SOD did not decrease at higher concentrations of this isoform (Fig. 2B).Figure 2:Effect of SOD on the cytotoxicity of SIN-1 to HepG2 cells. Panel A, effect of catalase and azide on the potentiation of SIN-1 cytotoxicity by Cu,Zn-SOD. Viability of the HepG2 cells was determined after a 24-h incubation with 0.316 mM SIN-1 in the presence of the indicated concentrations of Cu,Zn-SOD. Additions were as follows: SIN-1 (•), SIN-1 plus 0.316 mM azide (▴), SIN-1 plus catalase (200 units/ml) (f), SIN-1 plus boiled Cu,Zn-SOD (▴), Cu,Zn-SOD in the absence of SIN-1 (□). Results are mean ± S.E. from three experiments. Panel B, comparison of the effects of Cu,Zn-SOD (•) and Mn-SOD (E) on the cytotoxicity of SIN-1 to HepG2 cells. The cytotoxic effect of 0.316 mM SIN-1 was determined in the presence of the indicated concentrations of Cu,Zn-SOD or Mn-SOD. Viability was determined after a 24-h incubation period. Results are mean ± S.E. from three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Since SOD increases the rate of production of H2O2 by catalyzing O dismutation, the effect of catalase on the SIN-1 plus SOD toxicity was determined. Catalase (200 units/ml) abolished the SOD-mediated cytotoxic effect of SIN-1 at all Cu,Zn-SOD concentrations tested (Fig. 2A). The possible involvement of intracellular H2O2 and catalase in the cytotoxic effect of SOD plus SIN-1 was studied using sodium azide, an inhibitor of the intracellular catalase. Sodium azide slightly increased the toxicity of SIN-1 in the absence of SOD and further potentiated the cytotoxic effect produced by the combination of SIN-1 plus Cu,Zn-SOD (Fig. 2A).Since H2O2 can be a precursor for formation of OH, the possible involvement of OH-like species in the SOD-mediated cytotoxic effect of SIN-1 was evaluated using OH scavengers. Viability of the HepG2 cells was lowered by 10% by 0.632 mM SIN-1 and by 90% in the presence of SIN-1 plus 100 units/ml Cu,Zn-SOD (Fig. 3). Thiourea and uric acid were very effective in protecting the HepG2 cells against this SOD-mediated cytotoxicity. However, other OH scavengers tested, including Me2SO, sodium formate, and mannitol, had no protective effect (Fig. 3). Similarly, ethanol (10-100 mM) and benzoic acid (0.5-10 mM) also had no protective effect (data not shown).Figure 3:Effect of hydroxyl radical scavengers on the potentiation of SIN-1 cytotoxicity by Cu,Zn-SOD. Viability of the HepG2 cells was determined after a 24-h incubation without SIN-1 (C), with 0.632 mM SIN-1 (S), or with 0.632 mM SIN-1 plus 100 units/ml Cu,Zn-SOD (SS). All the other incubations contained SIN-1 plus Cu,Zn-SOD plus the indicated antioxidants at the following concentrations: thiourea, 0.1, 0.5, 1, and 5 mM, respectively; urate, 0.1, 0.5, and 1 mM, respectively; Me2SO, 0.1, 0.316, 1, and 3.16 mM, respectively; formate, 0.1, 0.316, 1, and 3.16 mM, respectively; mannitol, 1, 3.16, and 10 mM, respectively. Results are mean ± S.E. from four experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Formation of OH and other potent oxidizing species from H2O2 requires catalysis by transition metals such as iron. The role of transition metals in the SOD plus SIN-1 cytotoxicity was studied using the iron chelators deferoxamine and 2,2′-dipyridyl. As shown in Fig. 4, the cytotoxicity of SOD plus SIN-1 was reduced in a concentration-dependent manner by co-incubation of the samples with these two iron chelators. In contrast, EDTA and DTPA had only a small protective effect; the viability of the cells exposed to the SIN-1 plus SOD combination increased from 20% in the absence of chelator to 34 and 39% in the presence of 1 mM EDTA or 1 mM DTPA, respectively (data not shown).Figure 4:Effect of iron chelators on the potentiation of SIN-1 cytotoxicity by Cu,Zn-SOD. Experiments were carried out as described in the legend to Fig. 3: control (no SIN-1 added) (C), 0.632 mM SIN-1 (S), and 0.632 mM SIN-1 plus 100 units/ml Cu,Zn-SOD, (SS). All the other incubations contained SIN-1 plus Cu,Zn-SOD plus the indicated chelators at the following concentrations: deferoxamine, 0.01, 0.0316, 0.1, and 0.316 mM, respectively; 2,2′-dipyridyl, 0.01, 0.0316, 0.1, and 0.316 mM, respectively. Results are mean ± S.E. from three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The role of intracellular glutathione in the protection of HepG2 cells against the toxicity of SIN-1 itself or the potentiation of SIN-1 toxicity by SOD was studied using BSO, which inhibits GSH synthesis (38Griffith O.W. Meister A. J. Biol. Chem. 1979; 254: 7558-7560Abstract Full Text PDF PubMed Google Scholar). GSH functions to help remove H2O2 via glutathione peroxidase activity; GSH can also scavenge peroxynitrite (29Brunelli L. Crow J.P. Beckman J.S. Arch. Biochem. Biophys. 1995; 316: 327-334Crossref PubMed Scopus (262) Google Scholar). BSO at a concentration (0.1 mM) that depleted cellular GSH by 85 to 90% increased the cytotoxic effect of SIN-1 itself from about 5% loss of viability in the absence of BSO treatment to about 50% loss of viability after BSO treatment (Fig. 5). In addition, the BSO treatment also increased the potentiation of SIN-1 cytotoxicity produced by Cu,Zn-SOD (Fig. 5), analogous to the potentiation produced by azide (Fig. 2A).Figure 5:Effect of BSO on SIN-1/SOD cytotoxicity. HepG2 cells were treated without (open bars) or with 0.1 mM BSO (hatched bars) for 2 h, prior to the addition of 0.316 mM SIN-1 plus the indicated concentrations of Cu,Zn-SOD. Viability was determined after a 24-h incubation. Results are mean ± S.E. from three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Production of H2O2The protective effects of catalase and GSH against SIN-1 and especially SIN-1 plus SOD toxicity suggests that H2O2 was involved in the overall mechanism leading to toxicity. The increase in SIN-1 toxicity produced by addition of Cu,Zn-SOD or Mn-SOD could reflect elevated production of H2O2 in the presence of these enzymes. Accumulation of H2O2 from SIN-1 was therefore determined. Measurement of H2O2 by two independent methods showed agreement between the concentrations of accumulated H2O2 by the various reaction systems and the cytotoxicity to the HepG2 cells. For example, the concentration of H2O2 in the SIN-1-containing system increased in the presence of 100 units/ml Cu,Zn-SOD as well as in the presence of both 100 and 1000 units/ml Mn-SOD (Fig. 6); this accumulation of H2O2 is consistent with the enhancement of SIN-1 toxicity produced in the presence of these concentrations of SOD. Of interest was the observation that levels of H2O2 produced from SIN-1 were actually lower in the presence of higher concentrations of the Cu,Zn-SOD, but not the Mn-SOD (Fig. 6). The Cu,Zn-SOD potentiation of SIN-1 toxicity was also lower at elevated concentrations of the Cu,Zn-SOD (Fig. 2B). The possibility that catalase was present as a contaminant in the Cu,Zn-SOD preparation, thereby decreasing H2O2 accumulation and SIN-1 toxicity was ruled out by demonstrating that very high concentrations of azide (5 mM), which should completely inhibit any catalase that could be present, did not affect the accumulation of H2O2 produced from SIN-1 in the presence of low and high concentrations of the Cu,Zn-SOD (data not shown).Figure 6:Accumulation of H2O2 from the decomposition of SIN-1. The formation of H2O2 from 0.316 mM SIN-1 was determined by assaying for the oxidation of methanol by the catalase-H2O2 compound I complex as described under "Experimental Procedures." Experiments were carried out in the absence (blackbar) or presence of either 100 units/ml Cu,Zn-SOD or Mn-SOD (empty bars) or 1000 units/ml Cu,Zn-SOD or Mn-SOD (hatched bars). Results are mean ± S.E. from three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Spin Trapping of OHThe availability of H2O2 accumulated after a 24-h incubation in the SIN-1 plus SOD systems for iron (Fe(II))-catalyzed decomposition was demonstrated by spin trapping with DMPO (Fig. 7). The products of the Fenton reaction (OH radical or Fe(IV) species) were converted by reaction with Me2SO to methyl radicals which were then spin-trapped with DMPO. Only low levels of OH radicals (DMPO/CH3 adducts; aN = 16.24 G; aH = 23.19 G) could be detected (Fig. 7) upon addition of Fe(II) to the samples which were incubated with either SIN-1 alone (traceE) or SIN-1 plus 1000 units/ml Cu,Zn-SOD (traceB). Formation of OH was significantly higher in the samples incubated with SIN-1 plus 100 units/ml Cu,Zn-SOD, 100 units/ml Mn-SOD, or 1000 units/ml Mn-SOD (Fig. 7, tracesA, C, and D). Production of OH (DMPO/CH3 adduct) therefore paralleled accumulation of H2O2 (Fig. 6).Figure 7:T