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• W1982484092 abstract "We have investigated the ability of intracellular vitamin C to protect human umbilical vein endothelial cells from exposure to hypochlorous acid (HOCl) and a range of derived chloramines. Ascorbate provided minimal protection against the cytotoxicity induced by these oxidants, as measured by propidium iodide uptake. In contrast, there was a marked effect on apoptosis, monitored by caspase-3 activation and phosphatidylserine exposure. Extended incubation of the cells with glycine chloramine or histamine chloramine completely blocked apoptosis initiated in the cells by serum withdrawal. This effect was significantly abrogated by ascorbate. Inhibition of apoptosis required the oxidant to be present for an extended period after serum withdrawal and occurred prior to caspase-3 activation. General protection of thiols by ascorbate was not responsible for the protection of apoptosis, because intracellular oxidation by HOCl or chloramines was not prevented in supplemented cells. The results suggest a new role for vitamin C in the regulation of apoptosis. We propose that, by protection of an oxidant-sensitive step in the initiation phase, ascorbate allows apoptosis to proceed in endothelial cells under sustained oxidative stress. We have investigated the ability of intracellular vitamin C to protect human umbilical vein endothelial cells from exposure to hypochlorous acid (HOCl) and a range of derived chloramines. Ascorbate provided minimal protection against the cytotoxicity induced by these oxidants, as measured by propidium iodide uptake. In contrast, there was a marked effect on apoptosis, monitored by caspase-3 activation and phosphatidylserine exposure. Extended incubation of the cells with glycine chloramine or histamine chloramine completely blocked apoptosis initiated in the cells by serum withdrawal. This effect was significantly abrogated by ascorbate. Inhibition of apoptosis required the oxidant to be present for an extended period after serum withdrawal and occurred prior to caspase-3 activation. General protection of thiols by ascorbate was not responsible for the protection of apoptosis, because intracellular oxidation by HOCl or chloramines was not prevented in supplemented cells. The results suggest a new role for vitamin C in the regulation of apoptosis. We propose that, by protection of an oxidant-sensitive step in the initiation phase, ascorbate allows apoptosis to proceed in endothelial cells under sustained oxidative stress. hypochlorous acid 7-amino-4-methylcoumarin Asp-Glu-Val-Asp-7-amino-4-methylcoumarin Hanks' balanced salt solution human umbilical vein endothelial cells phosphatidylserine fluorescein isothiocyanate high performance liquid chromatography 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid reduced glutathione fluorescence-activated cell sorting chloramine Ascorbic acid (vitamin C) is an essential vitamin present in plant and animal cells (1Tsao C.S. Packer L. Fuchs J. Vitamin C in Health and Disease. Marcel Dekker, New York1997: 25-58Google Scholar, 2Smirnoff N. Curr. Opin. Plant Biol. 2000; 3: 229-235Crossref PubMed Google Scholar). Its functions are many, and relate mostly to its ability to act as a strong reducing agent (3Halliwell B. Whiteman M. Packer L. Fuchs J. Vitamin C in Health and Disease. Marcel Dekker, New York1997: 59-73Google Scholar, 4Frei B. England L. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6377-6381Crossref PubMed Scopus (1688) Google Scholar). Human plasma levels are typically 50–70 μm, whereas tissue concentrations range from around 1 mm for skeletal and smooth muscle to >10 mm in leukocytes, brain, adrenal glands, and lungs (1Tsao C.S. Packer L. Fuchs J. Vitamin C in Health and Disease. Marcel Dekker, New York1997: 25-58Google Scholar). Ascorbate is an essential cofactor for the enzymes responsible for the hydroxylation of proline and lysine and is, therefore, necessary for the synthesis of collagen (5Barnes M.J. Ann. N. Y. Acad. Sci. 1975; 258: 264-277Crossref PubMed Scopus (119) Google Scholar). It is also required for the synthesis of hormones and neurotransmitters in the adrenals and the brain, and its high levels in leukocytes suggest a vital role in the immune response (1Tsao C.S. Packer L. Fuchs J. Vitamin C in Health and Disease. Marcel Dekker, New York1997: 25-58Google Scholar, 6Goldschmidt M.C. Am. J. Clin. Nutr. 1991; 54: 1214S-1220SCrossref PubMed Scopus (41) Google Scholar). Ascorbate is highly water-soluble, and its ability to act as a one- or two-electron reductant for a wide variety of biological oxidizing species has led to a great deal of interest in its role as a major anti-oxidant. This role has been supported by many in vitrostudies demonstrating the ability of ascorbate to scavenge oxidants and radical species, including the vitamin E radical, peroxyl radicals, singlet oxygen, thiyl radicals, superoxide and peroxynitrite (3Halliwell B. Whiteman M. Packer L. Fuchs J. Vitamin C in Health and Disease. Marcel Dekker, New York1997: 59-73Google Scholar,7Bowry V.W. Ingold K.U. Stocker R. Biochem. J. 1992; 288: 341-344Crossref PubMed Scopus (462) Google Scholar, 8Hu M. Louie S. Cross C.E. Motchnik P. Halliwell B. J. Lab. Clin. Med. 1993; 121: 257-262PubMed Google Scholar, 9Berger T.M. Polidori M.C. Dabbagh A. Evans P.J. Halliwell B. Morrow J.D. Roberts L.J. Frei B. J. Biol. Chem. 1997; 272: 15656-15660Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), and to protect cells from the damaging reactions of exogenous oxidants such as H2O2, nitrogen dioxide, and X-irradiation (10Tu B. Wallin A. Moldeus P. Cotgreave I. Toxicology. 1995; 98: 125-136Crossref PubMed Scopus (17) Google Scholar, 11Witenberg B. Kletter Y. Kalir H.H. Raviv Z. Fenig E. Nagler A. Halperin D. Fabian I. Radiat. Res. 1999; 152: 468-478Crossref PubMed Scopus (52) Google Scholar, 12Alexandra E.K. Strom K. Cotgreave I.A. Biochem. Pharmacol. 1995; 50: 1339-1346Crossref PubMed Scopus (56) Google Scholar, 13Kanno S. Ishikawa M. Takayanagi M. Takayanagi Y. Sasaki K. Biol. Pharm. Bull. 2000; 23: 37-42Crossref PubMed Scopus (36) Google Scholar). Despite these studies, there is still debate about the importance of the anti-oxidant role in vivo (14Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. Oxford University Press, Oxford1999: 200-208Google Scholar). There is little direct evidence for anti-oxidant protection by ascorbate in vivo, and the symptoms of vitamin C deficiency (scurvy) appear to be more related to its hydroxylation reactions than to an anti-oxidant function (1Tsao C.S. Packer L. Fuchs J. Vitamin C in Health and Disease. Marcel Dekker, New York1997: 25-58Google Scholar). A significant oxidant for which ascorbate could provide effective protection is hypochlorous acid (HOCl).1 This is produced by stimulated phagocytic cells, via the action of myeloperoxidase, which uses H2O2 to catalyze the oxidation of halides, particularly Cl− (15Winterbourn C.C. Vissers M.C.M. Kettle A.J. Curr. Opin. Hematol. 2000; 7: 53-58Crossref PubMed Scopus (272) Google Scholar). HOCl is a powerful two-electron oxidant that reacts readily with a range of biological targets, and its production at sites of inflammation is associated with tissue and cell injury (16Prutz W.A. Arch. Biochem. Biophys. 1996; 332: 110-120Crossref PubMed Scopus (260) Google Scholar, 17Pullar J.M. Vissers M.C.M. Winterbourn C.C. IUBMB Life. 2000; 50: 259-266Crossref PubMed Scopus (226) Google Scholar). Direct evidence for this comes from studies that have detected myeloperoxidase and HOCl-modified proteins in atherosclerotic plaques, in the endothelial cells lining arterial vessel walls, and in inflammatory bowel disease (18Hazell L.J. Arnold L. Flowers D. Waeg G. Malle E. Stocker R. J. Clin. Invest. 1996; 97: 1535-1544Crossref PubMed Scopus (529) Google Scholar, 19Malle E. Waeg G. Schreiber R. Grone E.F. Sattler W. Grone H.J. Eur. J. Biochem. 2000; 267: 4495-4503Crossref PubMed Scopus (216) Google Scholar, 20McKenzie S.J. Baker M.S. Buffington G.D. Doe W.F. J. Clin. Invest. 1996; 98: 136-141Crossref PubMed Scopus (313) Google Scholar). HOCl reacts most readily with thiols, with a rate constant of >107m−1 s−1 at neutral pH (21Winterbourn C.C. Biochim. Biophys. Acta. 1985; 840: 204-210Crossref PubMed Scopus (412) Google Scholar, 22Folkes L.K. Candeias L.P. Wardman P. Arch. Biochem. Biophys. 1995; 323: 120-126Crossref PubMed Scopus (286) Google Scholar, 23Prutz W.A. Arch. Biochem. Biophys. 1999; 371: 107-114Crossref PubMed Scopus (56) Google Scholar). We, and others have shown that glutathione and protein thiols are oxidized in cells exposed to low concentrations of HOCl (24Tatsumi T. Fliss H. Am. J. Physiol. 1994; 267: H1597-H1607PubMed Google Scholar, 25Pullar J.M. Winterbourn C.C. Vissers M.C.M. Am. J. Physiol. 1999; 277: H1505-H1512PubMed Google Scholar, 26Pullar J.M. Vissers M.C.M. Winterbourn C.C. J. Biol. Chem. 2001; 276: 22120-22125Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Amino groups are also readily oxidized, with the generation of chloramine derivatives (-NHCl) that retain the Cl+-oxidizing equivalent of HOCl and extend its toxicity (27Samuels M.P. Warner J.O. Arch. Dis. Child. 1987; 62: 1099-1101Crossref PubMed Scopus (7) Google Scholar, 28Prutz W.A. Arch. Biochem. Biophys. 1998; 349: 183-191Crossref PubMed Scopus (107) Google Scholar). Chloramines also react readily with thiols and are variably cytotoxic, depending on their ability to cross cell membranes (27Samuels M.P. Warner J.O. Arch. Dis. Child. 1987; 62: 1099-1101Crossref PubMed Scopus (7) Google Scholar, 29Peskin A.V. Winterbourn C.C. Free Radic. Biol. Med. 2001; 30: 572-579Crossref PubMed Scopus (290) Google Scholar,30Thomas E.L. Jefferson M.M. Learn D.B. King C.C. Dabbous M.K. Redox Rep. 2000; 5: 191-196Crossref PubMed Scopus (14) Google Scholar). Scavenging of HOCl and chloramines by cell thiols and methionine, together with enzymatic regeneration of these groups, is a likely defense against their toxicity (31Levine R.L. Moskovitz J. Stadtman E.R. IUBMB Life. 2000; 50: 301-307Crossref PubMed Scopus (325) Google Scholar). However, both HOCl and chloramines react with ascorbate, the former with a reaction rate constant of approx 5 × 106m−1s−1 (22Folkes L.K. Candeias L.P. Wardman P. Arch. Biochem. Biophys. 1995; 323: 120-126Crossref PubMed Scopus (286) Google Scholar, 29Peskin A.V. Winterbourn C.C. Free Radic. Biol. Med. 2001; 30: 572-579Crossref PubMed Scopus (290) Google Scholar, 32Carr A.C. Hawkins C.L. Thomas S.R. Stocker R. Frei B. Free Radic. Biol Med. 2001; 30: 526-536Crossref PubMed Scopus (78) Google Scholar). Ascorbate has also been shown to protect protein targets and low density lipoprotein from reaction with these chlorinated oxidants and may be particularly effective against chloramine-mediated injury (14Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. Oxford University Press, Oxford1999: 200-208Google Scholar, 32Carr A.C. Hawkins C.L. Thomas S.R. Stocker R. Frei B. Free Radic. Biol Med. 2001; 30: 526-536Crossref PubMed Scopus (78) Google Scholar, 33Ischiropoulos H. Arch. Biochem. Biophys. 1998; 356: 1-11Crossref PubMed Scopus (918) Google Scholar). We have assessed the effect of intracellular ascorbate on the toxicity of HOCl and a range of chloramines, using cultured human umbilical vein endothelial cells (HUVEC) as a model system. We have shown that HOCl can oxidize susceptible thiol-containing enzymes and glutathione and initiate apoptosis in these cells (25Pullar J.M. Winterbourn C.C. Vissers M.C.M. Am. J. Physiol. 1999; 277: H1505-H1512PubMed Google Scholar, 34Vissers M.C.M. Pullar J.M. Hampton M.B. Biochem. J. 1999; 344: 443-449Crossref PubMed Scopus (92) Google Scholar). Sub-lethal concentrations of HOCl also initiate signaling responses and growth arrest in HUVEC and fibroblasts (34Vissers M.C.M. Pullar J.M. Hampton M.B. Biochem. J. 1999; 344: 443-449Crossref PubMed Scopus (92) Google Scholar, 35Vile G.F. Rothwell L.A. Kettle A.J. Arch. Biochem. Biophys. 1998; 359: 51-56Crossref PubMed Scopus (44) Google Scholar, 36Vile G.F. Rothwell L.A. Kettle A.J. Arch. Biochem. Biophys. 2000; 377: 122-128Crossref PubMed Scopus (44) Google Scholar). Using this model, it is therefore possible to investigate the effect of ascorbate on a range of oxidant-mediated responses. Under routine culture conditions HUVEC, and any other human cell line, are completely deficient in ascorbate (12Alexandra E.K. Strom K. Cotgreave I.A. Biochem. Pharmacol. 1995; 50: 1339-1346Crossref PubMed Scopus (56) Google Scholar,37Leist M. Raab B. Maurer S. Rosick U. Brigelius-Flohe R. Free Radic. Biol. Med. 2001; 21: 297-306Crossref Scopus (145) Google Scholar). Intracellular levels can be boosted by uptake as the reduced vitamin from supplemented medium (12Alexandra E.K. Strom K. Cotgreave I.A. Biochem. Pharmacol. 1995; 50: 1339-1346Crossref PubMed Scopus (56) Google Scholar). In the course of this study we found that, although cellular ascorbate offered only marginal protection of endothelial cells against the toxicity of HOCl and chloramines, there was a marked effect on apoptosis. Chlorinated oxidants were able to inhibit apoptosis, and this inhibition was completely abrogated by ascorbate. The ability of ascorbate to allow cells under oxidative stress to undergo apoptosis is an unexpected finding that may represent a new and specific function of vitamin C. All cell culture media and supplements were from Life Technologies, Inc., supplied by Invitrogen (Auckland, New Zealand). PS exposure was measured by the binding of Annexin V-FITC using the Apotest FITC kit from Nexins Research B.V. (Hoeven, The Netherlands). The substrate for caspase-3-like enzymes, Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC) was from Bachem, (Bubendorf, Switzerland). HOCl was from Reckitt and Coleman Ltd. (Auckland, New Zealand). Solutions were diluted into Hanks' balanced salt solution (HBSS; 10 mm phosphate buffer, pH 7.4, containing 1 mm CaCl2, 0.5 mmMgCl2, and 1 mg/ml glucose) and standardized by the absorbance at 292 nm at pH > 9 (ε = 350m−1 cm−1) (38Gazda M. Margerum D.W. Inorg. Chem. 1994; 33: 118-123Crossref Scopus (91) Google Scholar). Chloramine derivatives of NH3 and glycine were generated by mixing equal volumes of 10 mm amine solutions at pH 7.4 with 2 mm HOCl at 0–4 °C. With histamine, 4 mmsolutions were mixed with 2 mm HOCl to minimize the final concentration of histamine. The concentrations of chloramine were determined by reaction with 5-thio-2-nitrobenzoic acid, and the change in absorbance at 412 nm was monitored (ε = 14,140m−1 cm−1) (39Vissers M.C.M. Fantone J.C. Free Radic. Biol. Med. 1990; 8: 331-337Crossref PubMed Scopus (27) Google Scholar). All other reagents and chemicals were from Sigma Chemical Co. and were reagent grade or better. HUVEC were isolated from human umbilical cords by collagenase extraction and cultured in Medium 199 supplemented with 20% fetal calf serum and endothelial cell growth supplement until the second or third passage as previously described (34Vissers M.C.M. Pullar J.M. Hampton M.B. Biochem. J. 1999; 344: 443-449Crossref PubMed Scopus (92) Google Scholar). Cells were finally cultured in 24-well plates and used when confluent (approximately 120,000 cells per well). HUVEC were loaded with vitamin C by supplementing the medium with 1 mm sodium ascorbate for 18 h (12Alexandra E.K. Strom K. Cotgreave I.A. Biochem. Pharmacol. 1995; 50: 1339-1346Crossref PubMed Scopus (56) Google Scholar). After incubation with glycine-NHCl, 2 mm methionine was added to scavenge any remaining oxidant, and HUVEC were washed three times with HBSS and harvested by trypsin digestion. Cell pellets were stored at −80 °C until assay, when 150 μl of 0.27 m perchloric acid was added. Samples were centrifuged at 10,000 rpm for 2 min, and the ascorbate was measured in the supernatant by HPLC using an Aqua C18 5 micron column and electrochemical detection (40Lee W. Hamernyik P. Hutchinson M. Raisys V.A. Labbe R.F. Clin. Chem. 1982; 28: 2165-2169Crossref PubMed Scopus (81) Google Scholar). Ascorbate concentration was calculated from a standard curve of between 0 and 10 ng/ml run with each experiment. Prior to treatment with HOCl or chloramines, the HUVEC were washed three times to remove any potential oxidant scavengers and the medium was replaced with HBSS. Diluted solutions of oxidant were added to each well to give a final volume of 1 ml. When the cells were exposed to HOCl, the HBSS was replaced with fresh complete medium after 10 min. When the longer-lived chloramines were added, the cells were maintained in HBSS for the duration of oxidant exposure. Throughout the experiment the plates were incubated at 37 °C in 5% CO2. The effect of oxidant exposure on HUVEC was determined by dual staining with Annexin V-FITC and propidium iodide. Annexin V binding to PS on the cell surface provides an indicator for apoptotic cells, whereas necrotic cells take up propidium iodide and bind Annexin V due to a loss in membrane permeability (34Vissers M.C.M. Pullar J.M. Hampton M.B. Biochem. J. 1999; 344: 443-449Crossref PubMed Scopus (92) Google Scholar, 41Vermes I. Haanen C. Steffens-Nakken H. Reutelingsperger C. J. Immunol. Methods. 1995; 184: 39-51Crossref PubMed Scopus (4518) Google Scholar). After exposure to oxidants the cells were harvested from the wells by trypsin digestion. Attached cells were pooled with any detached cells from the supernatant and pelleted by centrifugation. Annexin V and propidium iodide were added according to the manufacturer's instructions, and sample fluorescence of 10,000 cells was analyzed in a bivariate flow cytometer. The HUVEC from three wells (approx 360,000 cells) were harvested by trypsin digestion and pooled. The cells were spun and pellets frozen at −80 °C. Immediately before assay the samples were thawed by the addition of 100 μl of buffer (100 mm Hepes with 10% (w/v) sucrose, 1% (w/v) CHAPS, 5 mm dithiothreitol, 10−4% (v/v) Nonidet P-40, 50 μm DEVD-AMC), and the rate of change in fluorescence was monitored at 370(ex) and 445(em) (42Stridh H. Kimland M. Jones D.P. Orrenius S. Hampton M.B. FEBS Lett. 1998; 429: 351-355Crossref PubMed Scopus (240) Google Scholar). The amount of AMC liberated was calculated from a standard curve. Reduced glutathione was measured by reaction with the cell-permeable reagent monobromobimane and detection by HPLC (43Vissers M.C.M. Winterbourn C.C. Biochem. J. 1995; 307: 57-62Crossref PubMed Scopus (112) Google Scholar). HUVEC were exposed to oxidants in 1 ml of HBSS. After a given time 20 μl of methionine (2 mm) was added to scavenge any remaining oxidant. The well volume was adjusted to 0.4 ml by carefully removing 0.6 ml, and 20 μl of monobromobimane (10 mm in acetonitrile) and 6 μl NaOH (1 mm) were added. After 10 min in the dark, trichloroacetic acid was added to a final concentration of 2.5% (v/v), and the wells were scraped with a rubber policeman. The cell debris was pelleted, and the GSH were analyzed by HPLC using a Brownlee Spheri5 ODS column and fluorescence detection at 394(ex) and 408(em). The concentration of GSH was determined from a standard curve of 0–4 μm run with each experiment. Similar to previous reports (12Alexandra E.K. Strom K. Cotgreave I.A. Biochem. Pharmacol. 1995; 50: 1339-1346Crossref PubMed Scopus (56) Google Scholar), overnight incubation of HUVEC with ascorbate resulted in its uptake in a concentration-dependent manner (Fig. 1). We routinely used 1 mmascorbate in the preincubation medium, resulting in uptake of approximately 0.9 nmol per 120,000 cells. This amount corresponds to around 50% of the GSH in these cells (2.05 ± 0.19 nmol per 120,000 cells). Given that the intracellular concentration of GSH is on the order of 2–5 mm (14Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. Oxford University Press, Oxford1999: 200-208Google Scholar), the ascorbate concentration approximates in vivo levels of 1–2 mm (1Tsao C.S. Packer L. Fuchs J. Vitamin C in Health and Disease. Marcel Dekker, New York1997: 25-58Google Scholar). HOCl and the chloramine derivatives of NH3, glycine, and histamine were variably cytotoxic to HUVEC, as measured by propidium iodide uptake (Fig. 2). HOCl and NH2Cl caused almost complete necrosis at 100 and 50 μm, respectively, whereas glycine-NHCl and histamine-NHCl were less toxic. This toxicity relates directly to the ability of the oxidants to react with the cells. HOCl and NH2Cl are freely permeable (43Vissers M.C.M. Winterbourn C.C. Biochem. J. 1995; 307: 57-62Crossref PubMed Scopus (112) Google Scholar, 44Birdwell C.R. Gospodarowicz D. Nicholson G.L. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 3273-3277Crossref PubMed Scopus (182) Google Scholar) and were rapidly consumed, whereas glycine-NHCl and histamine-NHCl remained present in the medium for a considerable time (Fig. 3). When HUVEC were supplemented with ascorbate, there was no difference in the level of necrosis caused by exposure to HOCl or any of the chloramines (Fig. 2).Figure 3Consumption of HOCl and chloramines by HUVEC. Solutions (1 ml) of 25 μm HOCl (●), 25 μm NH2Cl (▪), 100 μmglycine-NHCl (▴), or 50 μm histamine-NHCl (▾) were added to cells in HBSS, in 24-well plates. The supernatants were sampled at given times, and the remaining oxidant was assayed by reaction with 5-thio-2-nitrobenzoic acid. In control wells without cells all oxidants were stable for the duration of the experiment (not shown). Results are the means ± S.D. of at least three estimates.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We have previously shown that apoptosis occurs with doses of HOCl between 25 and 50 nmol/120,000 cells (34Vissers M.C.M. Pullar J.M. Hampton M.B. Biochem. J. 1999; 344: 443-449Crossref PubMed Scopus (92) Google Scholar). To determine whether ascorbate affected apoptosis, we measured activation of caspase-3-like enzymes and monitored PS exposure with Annexin V-FITC. When the cells were treated with 40 nmol of HOCl/well, supplementation with ascorbate increased caspase activity from 15.3 ± 2.7 pmol of AMC/min/360,000 cells in control cells to 18.2 ± 3.1 pmol of AMC/min/360,000 cells in supplemented cells (p < 0.05, n = 6). PS exposure appeared to be similarly increased, although the difference did not reach statistical significance (not shown). These studies with HUVEC maintained in serum suggested that ascorbate could enhance apoptosis in cells exposed to chlorinated oxidants. When the effect of ascorbate on apoptosis induced by chloramines was investigated, the cells were maintained in HBSS to prevent scavenging of the oxidant by the culture medium. We found that HBSS alone initiated apoptosis in a significant population of the cells as indicated by an increase in caspase activity and PS exposure (Fig. 4). Identical results were obtained when HUVEC were incubated in Medium 199 without fetal calf serum (not shown), suggesting that serum deprival was responsible for initiating the apoptotic process. The effect of chloramines on HUVEC apoptosis was investigated against this background activity. When HUVEC were exposed to NH2Cl we found that caspase activity was slightly increased, with or without ascorbate (Fig. 5). However, activation was progressively blocked when the cells were incubated with glycine-NHCl, with maximal inhibition at 40 or 50 μm. This inhibition was markedly decreased when the cells were supplemented with ascorbate (Fig. 5). The greatest difference was seen with 50 μm glycine-NHCl. At higher doses of oxidant caspase protection was less evident, although the enzyme activity was always higher in cells supplemented with ascorbate. Caspase activation was similarly inhibited by histamine-NHCl at doses between 10 and 40 μm, and ascorbate was maximally protective at around 25 μm (Fig. 5). This effect on the activation of caspase-3-like enzymes was also seen when PS exposure was monitored (Fig. 6and Table I). PS exposure in HUVEC exposed to 5 or 10 μm NH2Cl was similar to the HBSS control and was unaffected by ascorbate (Table I). In contrast, PS exposure was completely inhibited by 50 μmglycine-NHCl, and this inhibition was almost completely abrogated in cells supplemented with ascorbate (Fig. 6 and Table I). Glycine-NHCl prevented PS exposure with a concentration dependence identical to that required to block caspase activation and protection by ascorbate was similarly effective at oxidant doses between 30 and 75 μm. With 25 μm NH2Cl or 100 μm glycine-NHCl, where some necrosis was evident (Fig. 2) inhibition of PS exposure was not readily blocked by ascorbate.Table IEffect of chloramines and intracellular ascorbate on PS exposure in HUVEC initiated by serum withdrawalIncubation conditionsAmount of apoptotic cells (PS-positive, propidium iodide-negative)(n)No ascorbate+ Ascorbate%Medium 199 control5.0 ± 0.84.1 ± 0.9(4)HBSS27.5 ± 2.623.3 ± 3.8(6)NH2Cl 5 μm23.0 ± 2.026.0 ± 7.0(2) 10 μm21.5 ± 1.523.5 ± 1.5(2) 25 μm6.0 ± 1.03.2 ± 1.8(2)Glycine-NHCl 25 μm16.8 ± 1.918.8 ± 3.8(4) 50 μm6.7 ± 1.61-ap < 0.005 as determined by paired Student's t test for samples without and with ascorbate. No other sample pairs were significantly different.16.8 ± 1.01-ap < 0.005 as determined by paired Student's t test for samples without and with ascorbate. No other sample pairs were significantly different.(6) 100 μm4.3 ± 1.25.3 ± 2.3(4)Cells were incubated with various doses of NH2Cl or glycine-NHCl in HBSS for 4 h prior to assay with Annexin V as in Fig. 5.1-a p < 0.005 as determined by paired Student's t test for samples without and with ascorbate. No other sample pairs were significantly different. Open table in a new tab Cells were incubated with various doses of NH2Cl or glycine-NHCl in HBSS for 4 h prior to assay with Annexin V as in Fig. 5. In contrast with HOCl and NH2Cl, which are rapidly consumed by the cells and are undetectable after 20–60 min, glycine-NHCl and histamine-NHCl persist in the medium for the duration of the experiment. We therefore considered the possibility that inhibition of apoptosis by glycine-NHCl or histamine-NHCl was dependent on their being present at a particular time in the initiation or execution phases of apoptosis. To investigate this further, we varied the time of addition of glycine-NHCl. When 50 μm glycine-NHCl was present for the first 60 min of the incubation, then removed and replaced with fresh HBSS, there was an increase in caspase activity rather than a decrease (Fig. 7), indicating that under these conditions glycine-NHCl causes additional apoptosis. However, when the oxidant was added at 2 h after serum withdrawal there was marked and consistent inhibition of caspase activation that was completely prevented by ascorbate (Fig. 7) and that resembled the effect seen when glycine-NHCl was present throughout the 4-h incubation. When glycine-NHCl was added 30 min prior to harvesting, there was a decrease in caspase activity that was only partly prevented or reversed by ascorbate (Fig. 7). Low concentrations of HOCl added 2 h after serum withdrawal also prevented caspase activation, and this was significantly less in the presence of ascorbate (Fig. 7). Because HOCl is very short-lived in the medium, these results suggest that chlorinated oxidants can prevent apoptosis in serum-deprived cells prior to activation of caspase-3 and PS exposure and that this inhibition can be prevented or repaired by ascorbate. Given that thiols are favored targets for HOCl and chloramines (21Winterbourn C.C. Biochim. Biophys. Acta. 1985; 840: 204-210Crossref PubMed Scopus (412) Google Scholar, 23Prutz W.A. Arch. Biochem. Biophys. 1999; 371: 107-114Crossref PubMed Scopus (56) Google Scholar, 29Peskin A.V. Winterbourn C.C. Free Radic. Biol. Med. 2001; 30: 572-579Crossref PubMed Scopus (290) Google Scholar), we investigated whether ascorbate was able to prevent GSH oxidation. Supplementation of HUVEC with ascorbate did not alter the extent of GSH loss with increasing doses of HOCl, histamine-NHCl, or glycine-NHCl (Table II).Table IIEffect of HOCl and chloramines on HUVEC GSH content in the presence and absence of ascorbateOxidantConcentrationGSH levelsNo ascorbateWith ascorbate% controlHOCl 30 μm84 ± 890 ± 25 50 μm66 ± 1073 ± 14 100 μm27 ± 634 ± 7Glycine-NHCl 50 μm58 ± 563 ± 6Histamine-NHCl 50 μm61 ± 768 ± 11 100 μm38 ± 140 ± 2Control HUVEC contained 2.05 ± 0.19 nmol of GSH/120,000 cells and 2.27 ± 0.47 nmol/120,000 cells in ascorbate supplemented cells. The results shown are from a minimum of four estimates. Cells were analyzed 30 min after exposure to oxidants. There was no significant difference in the GSH loss in cells with or without ascorbate. Open table in a new tab Control HUVEC contained 2.05 ± 0.19 nmol of GSH/120,000 cells and 2.27 ± 0.47 nmol/120,000 cells in ascorbate supplemented cells. The results shown are from a minimum of four estimates. Cells were analyzed 30 min after exposure to oxidants. There was no significant difference in the GSH loss in cells with or without ascorbate. After a 2-h incubation the ascorbate levels in control cells had decreased slightly (Table III). This is likely to be due to export from the cells. When the cells were incubated with 25–100 μm glycine-NHCl, there was a substantially greater loss of ascorbate (Table III). This does not reflect leakage from the cells, because there was minimal cell lysis in this time (refer to Fig. 2).Table IIIAscorbate content of HUVEC after incubation with glycine-NHClnmol of ascorbate/120,000 cellsControl cells, t = 01.12 ± 0.14Control cells,t = 2 h in HBSS0.75 ± 0.26Glycine-NHCl 2 h, 50 μm0.32 ± 0.22 2 h, 75 μm0.19 ± 0.21 2 h, 100 μm0.24 ± 0.03Cellular ascorbate was measured before and after 2-h incubation in HBSS without and with glycine-NHCl. The results shown are means ± S.D. of four estimates. Open table in a new tab Cellular ascorbate was measured before and after 2-h incubation in HBSS without and with glycine-NHCl. The results shown are means ± S.D. of four estimates. In this study we have found that chlorinated oxidants are able to block apoptosis in serum-deprived HUVEC and that vitamin C is able to protect against this oxidative stress and allow progression of the apoptotic process. Both PS exposure and activation of caspase-3-like enzymes were similarly affected, suggesting that this inhibition occurred at a point central to the apoptotic program. The ability of chlorinated oxidants to inhibit apoptosis depended on their being present 2 h after serum withdrawal. Glycine-NHCl and histamine-NHCl reacted slowly with the cells and persisted in the medium for several hours, explaining their inhibitory effect. In contrast, HOCl and NH2Cl were rapidly consumed by the cells and inhibited apoptosis only when added at a later time. Although HOCl reacts rapidly with cells, it is likely to generate some chloramines that could affect apoptosis signaling. In fact, generation of these long-lived species and their scavenging by ascorbate could explain the slightly increased apoptosis in HOCl-treated supplemented cells. Our finding that ascorbate can protect the apoptotic machinery from this oxidant attack may represent a new and potentially important role for vitamin C. Although there are many situations when apoptosis may be an undesirable outcome of oxidant exposure or cell damage, it is also central to the clearance of damaged and depleted cells in a way that does not disturb the surrounding tissue. In contrast, necrosis promotes inflammation and is to be avoided. In many in vivosituations an oxidant stress could persist over an extended time and may result in oxidative inhibition of apoptosis. Our results suggest that ascorbate provides specific protection under these conditions, allowing apoptosis to proceed. Neutrophils and phagocytic cells accumulate very high concentrations of ascorbate (1Tsao C.S. Packer L. Fuchs J. Vitamin C in Health and Disease. Marcel Dekker, New York1997: 25-58Google Scholar). These cells are required to undergo apoptosis at sites of inflammation (45Fadeel B. Åhlin A. Henter J. Orrenius S. Hampton M.B. Blood. 1998; 92: 4808-4818Crossref PubMed Google Scholar, 46Savill J.S. Wyllie A.H. Henson J.E. Walport M.J. Henson P.M. Haslett C. J. Clin. Invest. 1997; 83: 865-875Crossref Scopus (1333) Google Scholar), where they themselves generate considerable amounts of chlorinated oxidants (16Prutz W.A. Arch. Biochem. Biophys. 1996; 332: 110-120Crossref PubMed Scopus (260) Google Scholar, 17Pullar J.M. Vissers M.C.M. Winterbourn C.C. IUBMB Life. 2000; 50: 259-266Crossref PubMed Scopus (226) Google Scholar, 47Klebanoff S.J. Proc. Assoc. Am Physicians. 1999; 111: 383-389Crossref PubMed Scopus (323) Google Scholar). Although their intracellular ascorbate has been proposed to provide a general protection against these oxidants (6Goldschmidt M.C. Am. J. Clin. Nutr. 1991; 54: 1214S-1220SCrossref PubMed Scopus (41) Google Scholar, 14Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. Oxford University Press, Oxford1999: 200-208Google Scholar, 48Winterbourn C.C. Vissers M.C.M. Biochim. Biophys. Acta. 1983; 763: 175-179Crossref PubMed Scopus (33) Google Scholar), it is tempting to speculate that a more vital function could be the protection of apoptosis that will allow successful resolution of inflammation. The consequences of oxidative inhibition of apoptosis would be either the survival of damaged cells or necrosis, and either outcome is undesirable. Our endothelial cell model may also have relevance for the pathology of atherosclerosis. Chlorinated oxidants are produced in the course of this disease, because myeloperoxidase and hypochlorite-modified proteins have been detected both in the plaque and the adjacent endothelial cells (18Hazell L.J. Arnold L. Flowers D. Waeg G. Malle E. Stocker R. J. Clin. Invest. 1996; 97: 1535-1544Crossref PubMed Scopus (529) Google Scholar, 19Malle E. Waeg G. Schreiber R. Grone E.F. Sattler W. Grone H.J. Eur. J. Biochem. 2000; 267: 4495-4503Crossref PubMed Scopus (216) Google Scholar). Whether apoptosis of these cells is involved in plaque development is not known, but the protection of this process in an oxidative environment could prevent necrosis or induce the persistence of dysfunctional endothelial cells. Apoptosis can be induced in cells under a variety of conditions, and several signaling pathways have been described (49Chandra J. Samali A. Orrenius S. Free Radic. Biol. Med. 2000; 29: 323-333Crossref PubMed Scopus (1111) Google Scholar, 50Fadeel B. Orrenius S. Zhivotovsky B. Leukemia. 2000; 14: 1514-1525Crossref PubMed Scopus (91) Google Scholar). The majority of agents cause mitochondrial disruption, and the release of cytochromec that binds to Apaf-1 in the cytoplasm. These events culminate in activation of the effector caspases, including caspase-3 that cleaves a number of structural and regulatory proteins (49Chandra J. Samali A. Orrenius S. Free Radic. Biol. Med. 2000; 29: 323-333Crossref PubMed Scopus (1111) Google Scholar, 50Fadeel B. Orrenius S. Zhivotovsky B. Leukemia. 2000; 14: 1514-1525Crossref PubMed Scopus (91) Google Scholar). Oxidant stress can initiate apoptosis, and this can be partly prevented by cellular anti-oxidants, including ascorbate (10Tu B. Wallin A. Moldeus P. Cotgreave I. Toxicology. 1995; 98: 125-136Crossref PubMed Scopus (17) Google Scholar, 11Witenberg B. Kletter Y. Kalir H.H. Raviv Z. Fenig E. Nagler A. Halperin D. Fabian I. Radiat. Res. 1999; 152: 468-478Crossref PubMed Scopus (52) Google Scholar). In our current experiments, exposure of HUVEC to NH2Cl or glycine-NHCl for 60 min resulted in an increase in apoptosis that was not greatly affected by ascorbate. These conditions are more similar to mostin vitro studies, in which the oxidant is delivered in a short initial exposure, and the cellular consequences, including apoptosis, are monitored for the ensuing period. Sustained oxidative stress can attenuate apoptosis by oxidative modulation of the caspase catalytic site, which has a critical thiol residue (49Chandra J. Samali A. Orrenius S. Free Radic. Biol. Med. 2000; 29: 323-333Crossref PubMed Scopus (1111) Google Scholar, 51Hampton M.B. Orrenius S. FEBS Lett. 1997; 414: 552-556Crossref PubMed Scopus (582) Google Scholar, 52Hampton M.B. Fadeel B. Orrenius S. Ann. N. Y. Acad. Sci. 1998; 854: 328-335Crossref PubMed Scopus (235) Google Scholar). H2O2 and the thiol reagent thiuram disulfide have been shown to affect caspase activity and processing by a thiol-dependent mechanism (51Hampton M.B. Orrenius S. FEBS Lett. 1997; 414: 552-556Crossref PubMed Scopus (582) Google Scholar,53Nobel C.S.I. Burgess D.H. Zhivotovsky B. Burkitt M.J. Orrenius S. Slater A.F.G. Chem. Res. Toxicol. 1997; 10: 636-643Crossref PubMed Scopus (126) Google Scholar). H2O2 treatment of Jurkat cells undergoing Fas-mediated apoptosis effectively delayed the activation of caspase-3, suggesting that repair of the oxidized thiol occurred once the H2O2 was consumed (51Hampton M.B. Orrenius S. FEBS Lett. 1997; 414: 552-556Crossref PubMed Scopus (582) Google Scholar). In another study (53Nobel C.S.I. Burgess D.H. Zhivotovsky B. Burkitt M.J. Orrenius S. Slater A.F.G. Chem. Res. Toxicol. 1997; 10: 636-643Crossref PubMed Scopus (126) Google Scholar), delayed exposure of HL60 and Jurkat cells to oxidized dithiocarbamates prevented apoptosis initiated by Fas ligand or etoposide. The oxidant-sensitive step in the initiation of apoptosis was proposed to be at a point immediately prior to the processing of caspase-3. These results are similar to our findings with chlorinated oxidants and would explain the timing of addition of the oxidant stress. Given the relative specificity of chlorinated oxidants for thiol groups, it is possible that inactivation of a caspase could explain the prevention of apoptosis, although the exact mechanism remains unclear. The more novel finding in our study, however, is that this effect of oxidants can be markedly prevented by ascorbate and suggests an intimate involvement of ascorbate in apoptosis signaling. We propose that ascorbate can protect or repair some component of the apoptotic cascade that allows activation of the effector caspases that commit the cell to apoptosis (Fig. 8). The ascorbate-mediated protection of apoptosis was marked at some doses of chlorinated oxidant, but was ineffective at higher levels of exposure. This could indicate that the anti-oxidant or repair capacity of ascorbate can be overwhelmed by higher oxidant concentrations, or that, at these levels that cause necrotic cell death, apoptosis cannot occur for other reasons, such as depletion of ATP (54Lelli J.L.J. Becks L.L. Dabrowska M.I. Hinshaw D.B. Free Radic. Biol. Med. 1998; 25: 694-702Crossref PubMed Scopus (133) Google Scholar, 55Richter C. Schweizer M. Cossarizza A. Franceschi C. FEBS Lett. 1996; 378: 107-110Crossref PubMed Scopus (444) Google Scholar). That ascorbate provides specific protection of a component of the apoptotic process is suggested by its comparative ineffectiveness in preventing GSH oxidation or in protecting the cells against the toxicity of HOCl or chloramines. Neither process was significantly affected by ascorbate, suggesting that inhibition of general thiol oxidation does not itself account for the effect on apoptosis. Given the relative reaction rate constants of HOCl and chloramines with ascorbate and thiols (23Prutz W.A. Arch. Biochem. Biophys. 1999; 371: 107-114Crossref PubMed Scopus (56) Google Scholar, 29Peskin A.V. Winterbourn C.C. Free Radic. Biol. Med. 2001; 30: 572-579Crossref PubMed Scopus (290) Google Scholar), it is predicted that the intracellular ascorbate concentration should exceed the thiol concentration by 10-fold for it to act as an effective scavenger. This is not the case, with the ascorbate concentration being half that of GSH and much less than the protein thiols in HUVEC (25Pullar J.M. Winterbourn C.C. Vissers M.C.M. Am. J. Physiol. 1999; 277: H1505-H1512PubMed Google Scholar). However, intracellular GSH also did not prevent chloramine-mediated ascorbate oxidation and did not appear to reduce oxidized ascorbate. Hence, the cellular environment is clearly more complex and reactions cannot be predicted from purely chemical considerations. In summary, our results show that intracellular ascorbate can specifically protect apoptosis in HUVEC in the presence of chlorinated oxidants. The anti-oxidant protection of a putative thiol-dependent step, critical for the progression of apoptosis, has not previously been described and may represent a vital function for this essential vitamin. Further experiments are required to determine the target of the oxidative and protective events, and we are currently investigating this phenomenon with other oxidants and cell types. We thank Mary Morrison for expert technical assistance with cell culture and caspase assays, Lisa Haring for FACS analysis, and Prof. Christine Winterbourn for critical reading of the manuscript." @default.
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• W1982484092 title "Regulation of Apoptosis by Vitamin C" @default.
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