Prostaglandins 431281-292, 1992
EFFECTS OF REACTIVE OXYGEN SPECIES ON EICOSANOID IN HUMAN ENDOTHELIAL CELLS
METABOLISM
I. Schimke', A. Griesmachd, G. Wei e12, H.G. Robhutted and W.M. Mull2 'Institute of Pathological and Clinical Biochemistry, 31nstitute of Biochemistry, Humboldt-Univereqy of Berlin, Schumann&r. 20/21, D-1040 Berlin, Germany; 2nd Dept. of Surgery, University of Vienna, Spitalgasse 23, A-1090 Vienna, Austria iABSTRACT The influence of reactive oxygen species was (Hz% used as model substance) on the formation and release of TXA2 cells PG12 and by cultured human endothelial Was In the presence of H2C12 concentrations which did analyzed. induce a general cell damage (analyzed by estimation not of the cellular concentration of energy rich phosphates and lipid formation of both extent of peroxidation), the a sigmoidal shape with respect to eicosanoids exhibited Increasing H202 concentration shortened the half time time. and TXC12 production. The maximum rates of PG12 and of PGIz separated by a the TXCIZ formation were delay of TXCIZ The ratio of PG12 and TXAZ formation was 100 to production. formation and l-2 to 1 at the 1 at the time of maximum PG12 maximum TXF12 formation. This effect of reactive time of oxygen species could,contribute to the reduction of the function of the endothelium in hemostasis and protective function vascular tone. Using antioxidants, the modulating of reactive oxygen species on the eicosanoid metabolism in endothelial cells was verified.
INTRODUCTION thromboxane fi2 Prostacyclin (PG12) and are (TXfi21 involved in the control of vascular tone and hemostasis via a blood-endothelium interactions. reciprocal function in Changes of the PG12/TXC12 ratio important role play an mellitus. in hypertension, atherosclerosis and diabetes cells produce TXA2 Since it is known that endothelial biobeside PGIz the control and modulation of the (1,21, have synthesis of the two eicosanoids in endothelial cells In recent years, some subject of investigations (31. been oxygen influence of reactive demonstrated an authors estabeen species on the eicosanoid metabolism. It has quantiblished that the cyclooxygenase requires catalytic Broties of fatty acid hydroperoxides as activator (4-61. (7) reported a self deactivation of this therton et al.
Copyright
0
1992
Butterworth-Heinemann
282
Prostagland.ins
during catalysis. another important phenomenon is the selective inhibition of the PGI2 synthetase by reactive oxygen species. In contrast to that, the TXfl2 synthetase is not influenced by comparable concentrations of such reactive molecules (E-101. Therefore, reactive oxygen species could change the ratio of PGI2 and TXC12 formation and thus diminish the protective function of endothelium in hemostasis and vascular tone. This view is in line with 5ome recent studies dealing with the influence of reactive oxygen species on the eicosanoid metabolism in cells. Whorton et al. (111 demonstrated a H202-induced dose-dependent inhibition of the PGI2 formation in cultured porcine aortic endothelial cells. In contrast, Harlan et al. found a (121 timeand dose-dependent increase of PGI2 release from bovine and humane endothelial cells exposed to reactive oxygen species. The TX02 formation wa5 not analyzed in these studies. Taylor et al. (13) demonstrated the influence of reactive oxygen species on the PGI2 and TXC12 formation in fibroblasts, but did not show the cellular and biochemical integrity of the cells during exposure to reactive oxygen species. It can be expected that high doses of those reactive molecules might influence the eicosanoid metabolism by a general cell damage in an unspecific manner. The aim of this study was to investigate the influence of (model substance for reactive oxygen species) on the Y&2 2 and TXCI2 biosynthesis and release of human endothelial cells. In order to exclude a general cell damage during the experiments, cellular fiTP and creatine phosphate (CP) concentrations as well as the extent of lipid peroxidation were measured. additionally, function of reactive the oxygen species in the modulation of the endothelial eicosametabolism was verified by experiments carried out noid the presence of inhibitors of eicosanoid formation and in scavangers for reactive oxygen species. e”Zyilll2
MCITERICIL CIND METHODS Cell culture were prepared using human umbilical Endothelial cells proveins. Cells were cultured according to the standard published cedure Additional modifications were (141. were subculture recently Only cells from the first (21. used in the experiments. The cells were identified as endothelial cells by the typical contact-inhibited morphology, factor VIII otherwise known as cobblestone (151, and by No contamination by myocytes (F VIII = vWF) staining (161. subculture. fibroblasts could be detected in the first experiments were carried out on cells cultured in 24 fK1 80000-90000 ce11s/cm2). well plate5 (average cell density: Hydroperoxide incubation these experiments, the For cell layers were gently the
cultur washed
medium with
was prewarmed
removed, (37OCl
Prostaglandins
buffered saline (containing 0.9 mtl Ca'+, 0.5 mti phy:phate and incubated with 300,ul and 5 mM glucose), of this Mg buffer containing different concentrations of H21J2 (Ex;,fz The influence of inhibitors of eicosanoid and mrll. radical metabolism was analyzed by preincubation of the such substances (preincubation time cells with and used concentrations o-f the substances are listed in results). procedure was Each experimental performed four-fold in triplicates. inhibitor OKY 046 was a generous The TXC12 synthetase gift of the ON0 Pharmaceutical Corp., Japan. All other substances were provided from commercial sources. Sample preparation and determination procedure FIfter the end of the incubation time, the supernatants were into micro-test tubes containing indomethacin transfered (30 ,uM final concentration) to avoid further formation of buffered eicosanoids. Cells were washed with phosphate saline. Cell lysates were prepared by adding of 250~1 of M HClO4 per well. Direct radioimmunoassays (BIOTECX) 0.5 were carried out in order to determine the concentration of the supernatant. There 6-keto-PGFlo and TXE12 in was no evidence of interference with the RI0s by incubation the buffer itself or any of other substances used. The speciand reliability of the assays for PG12 and TXA2 were fity confirmed by use of the cyclooxygenase inhibitor indomethainhibitor well as the TXfi2 synthetase OKY-046 as tin as reported recently (2). anaThe extent of lipid peroxidation after incubation was (17) via determination of lyzed according to Ohkawa et al. thiobarbituric acid reactive substances (TBORS). Cells and supernatants were jointly used as samples. In order to determine the intracellular FITP and CP content, the cell lysates were neutralized with 1M K2C03 and centriThe supernatants were analyzed for CITP and CITP plus fuged. by means of firefly luciferase (181. respectively, CP, Curve fitting for the time-dependent formation of eicosanoids fitting was carried out on the basis of a theoretical The from the observed experimental data. equation deduced numerical regression procedure published in the nonlinear fitting software package SIMFIT (191 was used. Statistical analysis Values are expressed as mean the Wilcoxon significances, were orProstaglandins
3.
4.
5.
6.
7.
El.
9.
10.
11.
12.
13.
14.
1s.
16.
291
Griesmacher, A., M. David and II-M. Muller. Freisetzung von Prostacyclin aus humanen Endothelzellen. 2. med. Lab.diagn. 30: 41, 1989. Kent, R-S., S.L. Diedrich and 0-R. Wharton. Regulation of vascular prostaglandin synthesis by metabolites of arachidonic acid in perfused rabbit aorta. J. Clin. Invest. 72: 455, 1983. Hemler, M.E. and W.E.M. Lands. Evidence for a peroxideinitiated free radical mechanism of prostaglandin biosynthesis. J. Biol. Chem. 2%: 6253, 1980. Lands, W.E.M., R.J. Kulmarcz and P.J. Marshall. Lipid peroxide actions in the regulation of prostacyclin biosynthesis. In: Free Radicals in Biology. (W.Fs. Pryor, ed.), Vol. VI, Academic Press, New York, 1984, p. 39. Brotherton, cI.F.fi. and J.C. Hoak. Prostacyclin biosynthesis in cultured vascular endothelium is limited by deactivation of cyclooxygenase. J. Clin. Invest. 72: 1255, 1983. Moncada, S., R.J. Gryglewski, S. Bunting and J.R. Vane. IJ lipid peroxide inhibits the enzyme in blood vessel microsomes that generates from prostaglandin endoperoxides the substance (prostaglandin Xl which prevent platelet aggregation. Prostaglandins 12: 715, 1976. Ham, E.CI., R.W. Egan, D.D. Soderman, P.H. Gale and Peroxidase dependent deactivation of F-c\. Kuehl. prostacyclin synthase. J. Biol. Chem. 258: 2191, 1978. Salomon, J.FI., D.R. Smith, R.J. Flower, S. Moncada and Further studies on the enzymatic conversion J.R. Vane. of prostaglandin endoperoxide into prostacyclin by porcin aorta microsomes. Biochim. Biophys. 0cta 523: 250, 1978. Whorton, A-R., M.E. Montgomery and R.S. Kent. Effect of hydrogen peroxide on prostaglandin and cellular integrity in cultured porcine aortic endothelial cells. J. Clin. Invest. 76: 295, 1985. Harlan, J.M. and K.S. Callahan. Role of hydrogen peroxide in the neutrophil-mediated release of prostacyclin from cultured endothelial cells. Invest. 74: 442, 1984. J. Clin. Taylor, L., M.J. Menconi and P. Polgar. Participation of hydroperoxides and oxygen radicals in the control of prostaglandin synthesis. J. Biol. Chem. 258: 6855, 1983. Jaffe, E-a., R.L. Nachman, C.G. Becker and C.R. Minck. Culture of human endothelial cells derived from umbilical vein. J. Clin. Invest. 52: 2745, 1973. Haudenschild, C-C., R.S. Contran, M-0. Gimbrone and J. Fine structure of vascular endothelium in Folkman. culture. J. Ultrastruct. Res. 50: 22, 1975. Jaffe, E.O., L.W. Hoyer and R.L. Nachman. Synthesis of antihemophilic factor antigen by cultured human endothelial cells. J. Clin. Invest. 52: 2757, 1973.
292
17.
18.
19.
20.
21. 22.
23.
24.
25.
26.
27.
28.
29. 30.
Prosraglandlns Ohkawa, H., N. Ohishi and K. Yagi. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. final. Biochem. 95: 351, 1979. Schopf, G., W. Mohl, J. Schuster and M.M. Mtiller. Effects of PICSO on purine nucleotides in ischemic and reperfused canine hearts. In: Coronary Sinus. (W. Mohl, E. Wolner and D. Glogar, eds.) Steinkopf-Verlag, Darmstadt, 1984, p. 508. Holzhtitter, H.G. and fi. Colosimo. SIMFIT = a microcomputer software-toolkit for modellistic studies in biology. Camp. clppl. Biol. Sci. 6_: 23, 1990. Halliwell, B. and J.M.C. Gutteridge. Oxygen toxicity, oxygen radicals, transition metals and deseases. qiochem. J. 219: 1, 1984. Schimke, 1. unpublished results. Yasuda, M. and T. Fujita. Effect of lipid peroxidation on phospholipase A2 activity of rat liver mitochondria. Jpn. J. Pharmacol. 27: 429, 1977. Sevarian, CI., R-6. Stein and J.F. Mead. Metabolism of epoxidized phosphatidylcholine by phospholipase A2 and epoxide hydrolase. Lipids a: 781, 1981. Nastainczyk, W., V. Ullrich, E. Cadenas and H. Sies. Mechanism of arachidonic acid-stimulated singlet oxygen formation by prostaglandin-endoperoxide synthase. In: Oxygen Radicals in Chemistry and Biology. (W. Elors, M. Walter de Gruyter Verlag, Saran and D. Tait, eds;.) Berlin, 1982, p. 441. Fujimoto, Y. and T. Fujita. Antioxidant effects on prostaglandin synthesis in rabbit kidney medulla slices. Experientia 38: 1472, 1982. Searle, A.J.F., C. Gee and R.L. Willson. Ellipticines and carbazoles as antioxidants. In: Oxygen Radicals in Chemistry and Biology. (W. Bors, M. Saran and D. Tait, eds.) Walter de Gruyter Verlag, Berlin, 1982, p. 377. Schimke, I., CI. Haberland, L. Will-Shahab and I. Kiittner. In vitro effect5 of reactive oxygen species on the P-receptor-adenylyl cyclase system. Mol. in press. Cell. Biochem. olkylperoxy-radicals. Bickel, E.C. and E.C. Kooyman. Part I. Reactions with 2:4:6_Trialkylphenols. J. Chem. Sot. m: 3211. Reactivity of thiols with asada, K. and S. Kanematsu. superoxide radicals. figric. Biol. Chem. 40: 1891 1976. Biochemistry of the SH group. Jocelyn, P.C. ncademic.
31.
flust,
metals Biol. 32.
in
oxygen
Med.
1: _
ions
19765,
3,
S-J.,
Rosen
ferrous
N-Y.
L-0.
Klebanoff, H.
33.
Press. S.D.,
1972.
Morehouse,
C.E.
radical
reactions.
Role of Free Radicals
J.
1985. 0-M.
Waltersdorph,
Oxygen-based and
Thomas.
free
deferoxamine.
B-R.
radical J.
Niche1
generation
Biol.
Chem.
and by
264:
1989.
Gutterridge. J.N.C., A. Richmond and 8. Halliwell. Inhibition of iron-catalysed formation of hydroxyl peroxidation by radicals from superoxide and lipid desferrioxamine. Biochem. J. m: 469, 1979.
Editor:
G.
Kaley
Received:
6-15-91
Accepted:
11-26-91
EFFECTS OF REACTIVE OXYGEN SPECIES ON EICOSANOID IN HUMAN ENDOTHELIAL CELLS
METABOLISM
I. Schimke', A. Griesmachd, G. Wei e12, H.G. Robhutted and W.M. Mull2 'Institute of Pathological and Clinical Biochemistry, 31nstitute of Biochemistry, Humboldt-Univereqy of Berlin, Schumann&r. 20/21, D-1040 Berlin, Germany; 2nd Dept. of Surgery, University of Vienna, Spitalgasse 23, A-1090 Vienna, Austria iABSTRACT The influence of reactive oxygen species was (Hz% used as model substance) on the formation and release of TXA2 cells PG12 and by cultured human endothelial Was In the presence of H2C12 concentrations which did analyzed. induce a general cell damage (analyzed by estimation not of the cellular concentration of energy rich phosphates and lipid formation of both extent of peroxidation), the a sigmoidal shape with respect to eicosanoids exhibited Increasing H202 concentration shortened the half time time. and TXC12 production. The maximum rates of PG12 and of PGIz separated by a the TXCIZ formation were delay of TXCIZ The ratio of PG12 and TXAZ formation was 100 to production. formation and l-2 to 1 at the 1 at the time of maximum PG12 maximum TXF12 formation. This effect of reactive time of oxygen species could,contribute to the reduction of the function of the endothelium in hemostasis and protective function vascular tone. Using antioxidants, the modulating of reactive oxygen species on the eicosanoid metabolism in endothelial cells was verified.
INTRODUCTION thromboxane fi2 Prostacyclin (PG12) and are (TXfi21 involved in the control of vascular tone and hemostasis via a blood-endothelium interactions. reciprocal function in Changes of the PG12/TXC12 ratio important role play an mellitus. in hypertension, atherosclerosis and diabetes cells produce TXA2 Since it is known that endothelial biobeside PGIz the control and modulation of the (1,21, have synthesis of the two eicosanoids in endothelial cells In recent years, some subject of investigations (31. been oxygen influence of reactive demonstrated an authors estabeen species on the eicosanoid metabolism. It has quantiblished that the cyclooxygenase requires catalytic Broties of fatty acid hydroperoxides as activator (4-61. (7) reported a self deactivation of this therton et al.
Copyright
0
1992
Butterworth-Heinemann
282
Prostagland.ins
during catalysis. another important phenomenon is the selective inhibition of the PGI2 synthetase by reactive oxygen species. In contrast to that, the TXfl2 synthetase is not influenced by comparable concentrations of such reactive molecules (E-101. Therefore, reactive oxygen species could change the ratio of PGI2 and TXC12 formation and thus diminish the protective function of endothelium in hemostasis and vascular tone. This view is in line with 5ome recent studies dealing with the influence of reactive oxygen species on the eicosanoid metabolism in cells. Whorton et al. (111 demonstrated a H202-induced dose-dependent inhibition of the PGI2 formation in cultured porcine aortic endothelial cells. In contrast, Harlan et al. found a (121 timeand dose-dependent increase of PGI2 release from bovine and humane endothelial cells exposed to reactive oxygen species. The TX02 formation wa5 not analyzed in these studies. Taylor et al. (13) demonstrated the influence of reactive oxygen species on the PGI2 and TXC12 formation in fibroblasts, but did not show the cellular and biochemical integrity of the cells during exposure to reactive oxygen species. It can be expected that high doses of those reactive molecules might influence the eicosanoid metabolism by a general cell damage in an unspecific manner. The aim of this study was to investigate the influence of (model substance for reactive oxygen species) on the Y&2 2 and TXCI2 biosynthesis and release of human endothelial cells. In order to exclude a general cell damage during the experiments, cellular fiTP and creatine phosphate (CP) concentrations as well as the extent of lipid peroxidation were measured. additionally, function of reactive the oxygen species in the modulation of the endothelial eicosametabolism was verified by experiments carried out noid the presence of inhibitors of eicosanoid formation and in scavangers for reactive oxygen species. e”Zyilll2
MCITERICIL CIND METHODS Cell culture were prepared using human umbilical Endothelial cells proveins. Cells were cultured according to the standard published cedure Additional modifications were (141. were subculture recently Only cells from the first (21. used in the experiments. The cells were identified as endothelial cells by the typical contact-inhibited morphology, factor VIII otherwise known as cobblestone (151, and by No contamination by myocytes (F VIII = vWF) staining (161. subculture. fibroblasts could be detected in the first experiments were carried out on cells cultured in 24 fK1 80000-90000 ce11s/cm2). well plate5 (average cell density: Hydroperoxide incubation these experiments, the For cell layers were gently the
cultur washed
medium with
was prewarmed
removed, (37OCl
Prostaglandins
buffered saline (containing 0.9 mtl Ca'+, 0.5 mti phy:phate and incubated with 300,ul and 5 mM glucose), of this Mg buffer containing different concentrations of H21J2 (Ex;,fz The influence of inhibitors of eicosanoid and mrll. radical metabolism was analyzed by preincubation of the such substances (preincubation time cells with and used concentrations o-f the substances are listed in results). procedure was Each experimental performed four-fold in triplicates. inhibitor OKY 046 was a generous The TXC12 synthetase gift of the ON0 Pharmaceutical Corp., Japan. All other substances were provided from commercial sources. Sample preparation and determination procedure FIfter the end of the incubation time, the supernatants were into micro-test tubes containing indomethacin transfered (30 ,uM final concentration) to avoid further formation of buffered eicosanoids. Cells were washed with phosphate saline. Cell lysates were prepared by adding of 250~1 of M HClO4 per well. Direct radioimmunoassays (BIOTECX) 0.5 were carried out in order to determine the concentration of the supernatant. There 6-keto-PGFlo and TXE12 in was no evidence of interference with the RI0s by incubation the buffer itself or any of other substances used. The speciand reliability of the assays for PG12 and TXA2 were fity confirmed by use of the cyclooxygenase inhibitor indomethainhibitor well as the TXfi2 synthetase OKY-046 as tin as reported recently (2). anaThe extent of lipid peroxidation after incubation was (17) via determination of lyzed according to Ohkawa et al. thiobarbituric acid reactive substances (TBORS). Cells and supernatants were jointly used as samples. In order to determine the intracellular FITP and CP content, the cell lysates were neutralized with 1M K2C03 and centriThe supernatants were analyzed for CITP and CITP plus fuged. by means of firefly luciferase (181. respectively, CP, Curve fitting for the time-dependent formation of eicosanoids fitting was carried out on the basis of a theoretical The from the observed experimental data. equation deduced numerical regression procedure published in the nonlinear fitting software package SIMFIT (191 was used. Statistical analysis Values are expressed as mean the Wilcoxon significances, were orProstaglandins
3.
4.
5.
6.
7.
El.
9.
10.
11.
12.
13.
14.
1s.
16.
291
Griesmacher, A., M. David and II-M. Muller. Freisetzung von Prostacyclin aus humanen Endothelzellen. 2. med. Lab.diagn. 30: 41, 1989. Kent, R-S., S.L. Diedrich and 0-R. Wharton. Regulation of vascular prostaglandin synthesis by metabolites of arachidonic acid in perfused rabbit aorta. J. Clin. Invest. 72: 455, 1983. Hemler, M.E. and W.E.M. Lands. Evidence for a peroxideinitiated free radical mechanism of prostaglandin biosynthesis. J. Biol. Chem. 2%: 6253, 1980. Lands, W.E.M., R.J. Kulmarcz and P.J. Marshall. Lipid peroxide actions in the regulation of prostacyclin biosynthesis. In: Free Radicals in Biology. (W.Fs. Pryor, ed.), Vol. VI, Academic Press, New York, 1984, p. 39. Brotherton, cI.F.fi. and J.C. Hoak. Prostacyclin biosynthesis in cultured vascular endothelium is limited by deactivation of cyclooxygenase. J. Clin. Invest. 72: 1255, 1983. Moncada, S., R.J. Gryglewski, S. Bunting and J.R. Vane. IJ lipid peroxide inhibits the enzyme in blood vessel microsomes that generates from prostaglandin endoperoxides the substance (prostaglandin Xl which prevent platelet aggregation. Prostaglandins 12: 715, 1976. Ham, E.CI., R.W. Egan, D.D. Soderman, P.H. Gale and Peroxidase dependent deactivation of F-c\. Kuehl. prostacyclin synthase. J. Biol. Chem. 258: 2191, 1978. Salomon, J.FI., D.R. Smith, R.J. Flower, S. Moncada and Further studies on the enzymatic conversion J.R. Vane. of prostaglandin endoperoxide into prostacyclin by porcin aorta microsomes. Biochim. Biophys. 0cta 523: 250, 1978. Whorton, A-R., M.E. Montgomery and R.S. Kent. Effect of hydrogen peroxide on prostaglandin and cellular integrity in cultured porcine aortic endothelial cells. J. Clin. Invest. 76: 295, 1985. Harlan, J.M. and K.S. Callahan. Role of hydrogen peroxide in the neutrophil-mediated release of prostacyclin from cultured endothelial cells. Invest. 74: 442, 1984. J. Clin. Taylor, L., M.J. Menconi and P. Polgar. Participation of hydroperoxides and oxygen radicals in the control of prostaglandin synthesis. J. Biol. Chem. 258: 6855, 1983. Jaffe, E-a., R.L. Nachman, C.G. Becker and C.R. Minck. Culture of human endothelial cells derived from umbilical vein. J. Clin. Invest. 52: 2745, 1973. Haudenschild, C-C., R.S. Contran, M-0. Gimbrone and J. Fine structure of vascular endothelium in Folkman. culture. J. Ultrastruct. Res. 50: 22, 1975. Jaffe, E.O., L.W. Hoyer and R.L. Nachman. Synthesis of antihemophilic factor antigen by cultured human endothelial cells. J. Clin. Invest. 52: 2757, 1973.
292
17.
18.
19.
20.
21. 22.
23.
24.
25.
26.
27.
28.
29. 30.
Prosraglandlns Ohkawa, H., N. Ohishi and K. Yagi. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. final. Biochem. 95: 351, 1979. Schopf, G., W. Mohl, J. Schuster and M.M. Mtiller. Effects of PICSO on purine nucleotides in ischemic and reperfused canine hearts. In: Coronary Sinus. (W. Mohl, E. Wolner and D. Glogar, eds.) Steinkopf-Verlag, Darmstadt, 1984, p. 508. Holzhtitter, H.G. and fi. Colosimo. SIMFIT = a microcomputer software-toolkit for modellistic studies in biology. Camp. clppl. Biol. Sci. 6_: 23, 1990. Halliwell, B. and J.M.C. Gutteridge. Oxygen toxicity, oxygen radicals, transition metals and deseases. qiochem. J. 219: 1, 1984. Schimke, 1. unpublished results. Yasuda, M. and T. Fujita. Effect of lipid peroxidation on phospholipase A2 activity of rat liver mitochondria. Jpn. J. Pharmacol. 27: 429, 1977. Sevarian, CI., R-6. Stein and J.F. Mead. Metabolism of epoxidized phosphatidylcholine by phospholipase A2 and epoxide hydrolase. Lipids a: 781, 1981. Nastainczyk, W., V. Ullrich, E. Cadenas and H. Sies. Mechanism of arachidonic acid-stimulated singlet oxygen formation by prostaglandin-endoperoxide synthase. In: Oxygen Radicals in Chemistry and Biology. (W. Elors, M. Walter de Gruyter Verlag, Saran and D. Tait, eds;.) Berlin, 1982, p. 441. Fujimoto, Y. and T. Fujita. Antioxidant effects on prostaglandin synthesis in rabbit kidney medulla slices. Experientia 38: 1472, 1982. Searle, A.J.F., C. Gee and R.L. Willson. Ellipticines and carbazoles as antioxidants. In: Oxygen Radicals in Chemistry and Biology. (W. Bors, M. Saran and D. Tait, eds.) Walter de Gruyter Verlag, Berlin, 1982, p. 377. Schimke, I., CI. Haberland, L. Will-Shahab and I. Kiittner. In vitro effect5 of reactive oxygen species on the P-receptor-adenylyl cyclase system. Mol. in press. Cell. Biochem. olkylperoxy-radicals. Bickel, E.C. and E.C. Kooyman. Part I. Reactions with 2:4:6_Trialkylphenols. J. Chem. Sot. m: 3211. Reactivity of thiols with asada, K. and S. Kanematsu. superoxide radicals. figric. Biol. Chem. 40: 1891 1976. Biochemistry of the SH group. Jocelyn, P.C. ncademic.
31.
flust,
metals Biol. 32.
in
oxygen
Med.
1: _
ions
19765,
3,
S-J.,
Rosen
ferrous
N-Y.
L-0.
Klebanoff, H.
33.
Press. S.D.,
1972.
Morehouse,
C.E.
radical
reactions.
Role of Free Radicals
J.
1985. 0-M.
Waltersdorph,
Oxygen-based and
Thomas.
free
deferoxamine.
B-R.
radical J.
Niche1
generation
Biol.
Chem.
and by
264:
1989.
Gutterridge. J.N.C., A. Richmond and 8. Halliwell. Inhibition of iron-catalysed formation of hydroxyl peroxidation by radicals from superoxide and lipid desferrioxamine. Biochem. J. m: 469, 1979.
Editor:
G.
Kaley
Received:
6-15-91
Accepted:
11-26-91
