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Effects of oxygen free radicals on human erythrocytes: protective effects of deferoxamine and allopurinol
Intracellular Generation of Free Radicals
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SULFHYDRYL OXIDATION BY PEROXYNITRITE. Rafael Radi, Joseph S. Beckman. Kenneth M. Bush and Bruce A. Freeman. Departments of Anesthesiology and Biochemistry, University of Alabama at Birmingham, Birmingham, AL 35233, U.S.A. Endothelial cells, macrophages, neutrophils and neuronal cells can generate 02- and -NO (endothelial-derived relaxation factor) at increased rates when activated or metabolically stressed. Reaction of 0 2 and .NO yields peroxynitrite anion (ONOO-), a potent oxidant which will oxidize both non-protein and protein sulfhydryls. At pH 7.4 and 37oC, apparent second order rate constants were 5900 M-l.s-1 and 1700 M I . s I for the reaction of peroxynitrite anion with free cysteine and the single thiol of albumin, respectively. These rate constants are three orders of magnitude greater than the corresponding rate constants for reaction of hydrogen peroxide with these sulfhydryls (pH 7.4). Unlike H202, which oxidizes thiolate anion, peroxynitrite anion reacted preferentially with the undissociated form of the thiol group. Peroxynitrite-dependent cysteine oxidation was 90% cyanide and DTT reducible, indicating that cystine was the main oxidation product. Electrophoretic analysis revealed that peroxynitrite did not result in disulfide-mediated dimerization or peptide cleavage of BSA at pH 7.4. Peroxynitrite oxidized the BSA-thiol group to an arsenite non-reducible product, while borohydride recovered 50% of the BSA-thiol, suggesting oxidation beyond sulfenic acid. Peroxynitrous acid was a less effective thiol oxidizing agent than its anion, and presumably the oxidation was mediated by its apparent decomposition products, i.e. hydroxyl radical and nitrogen dioxide. These observations show that secondary reactions in the vascular space between 02- and -NO can exert cytotoxic effects mediated, in part, by alteration of tissue sulfhydryl homeostasis.
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CYTOCHROME b 5 MEDIATED REDOX CYCLING OF ESTROGENS. Deodutta Roy, Henry W. Strobel 1, and Joachim G. Liehr. Dept. of Pharmacology & Toxicoio~gy, Univ. of Texas Med. Branch, Galveston, TX 77550 and Dept. of Biochemistry & MoL Biology, Univ. of Texas Health Sci. Ctr., Houston, TX 77225, USA.
Previously we have demonstrated microsomai cytochrome P 450catalyzed redox cycling of estrogens. In this study, we investigated the role of cytochrome b 5 ('o5) in redox cycling. Pure P 450c and peroxides oxidized diethylstilbestrol (DES) to diethylstilbestrol-4',4"-qulnone (DES Q). This oxidation by H20 2 was doubled by addition of b 5 to P 450c, but did not proceed with b 5 alone. The stimulation by b 5 of the P 450c-catalyzed oxidation of DES to DES O occurred via modulation of the Vmax of P 450c rather than of the Kin. DES O was reduced to DES by purified bs and NADH-dependent b5 reductase. Pretreatment of micrusomes with an antibody of b 5 reductase inhibited microsomal NADH-dependent reduction of DES O to DES. The b 5 likely participates in the oxidation of DES to DES O by interacting with P-450c and in the reduction of DES O to DES by interacting with b5 reductase. Thus, the study demonstrates that b5 plays an active role in biological oxidation and reduction reactions. Supported by NIH, NCI (CA 43232, CA 43233).
EFFECT~S OF OXYGEN FREE RADICALS ON HUMAN ERYTHR~: PROTECrlVE EFFECTS OF DEFEROXAMINE AND ALI/3PURINOL. Moheb A. Rashid, Olof Jonsson, Donald G.Roberts, and C45ran William-Olsson. Dept. of Thoracic&Cardiovascular Surgery,Sahlgrenska Hospital,413 45 Gothenburg,Sweden.
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Oxygen free radicals(OFR) are produced by blood trauma during cardiopulmonary bypass(CPB) in cardiac surgery leading to reduced blood cell theology and disturbed microcirculation.Deferoxamine(DFX) an iron chelator and allopurinol (ALLP) an antioxidant are OFR scavengers. Their protective effects on RFR induced red blood cell (RBC) damage in the presence of mechanical blood trauma was compared by estimating RBC filtrability(RFR) during 4 hours in 12 experiments using fresh human RBCs.Each exp. had 6 groups:-1: control; 2: hypoxa nthine (HX )&xa nthine oxida se (XOD ) 3:10 mic.g.DFX;4:10 mic.g.AI/~;5:HX&XOD+10 mic.g. DFX; 6 :HX&XOD+I 0 mic. g .ALLP. There was significant improvement of RFR in groups 3,4,5 and 6 compared to groups I&2(p<0.05).DFX showed significantly better RFR(42.4+5&40.0+1 mic.L/sec) than ALLP (24.7+2&22.2+4 mic_L/sec)Tp=0.00003&p=0.004 ) at l&2 h~urs respectively. In conclusion,both DFX and ALLP protected RBCs:DFX appears to enhance membrane stability and improve RFR,probably by neutralizing intracellular OFR. This should lead to further studies in cardiac surgical patients undergoing CPB.
SUBSTRATE INHIBITION OF XANTHINE OXlDASE AND ITS RELATIONSHIP WITH SUPEROXlDE RADICAL PRODUCTION. Homero Rubbo, Rafael Radi and Eugenio Prodanov. Departamento de Bioquimica. Facultad de Medicina, Universidad de la Republica. Montevideo, Uruguay. A possible relationship between intramolecular electron transport and substrate inhibition was studied in reactions catalyzed by xanthine oxidase. Using hypoxanthine and xanthine as substrates, the kinetics of product formation (uric acid and superoxide anion radical) were followed spectrophotometrically. Experiments with hypoxanthine showed an inhibition which fits an equation of the classical form: v = V.S/(K m +a.S + S2/Ki), with reaction velocity decreasing with increasing substrate concentration, from 1.0 to 9.3 x 10-4 M. With xanthine, the type of inhibition was more complex, perhaps because inactivation only occurs at very high xanthine concentrations. With both substrates, inhibition was not reverted by addition of SOD and/or catalase. The percent of monovalent electron flux to oxygen diminished by increasing substrate concentration in the range of 0.5 to 6.0 x 10-5 M xanthine or hypoxanthine. Over this range of substrate concentrations, increasing substrate concentration increased the percent of monovalent flux and at the same time diminishing the velocity of uric acid production. This effect was pH-dependent, more significant at pH 9.5 than pH 8.3, and more so for hypoxanthine than with xanthine. It is concluded that the alteration of the internal electron transport by high substrate concentrations favors the monovalent pathway and interferes with enzyme catalytic activity.
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SULFHYDRYL OXIDATION BY PEROXYNITRITE. Rafael Radi, Joseph S. Beckman. Kenneth M. Bush and Bruce A. Freeman. Departments of Anesthesiology and Biochemistry, University of Alabama at Birmingham, Birmingham, AL 35233, U.S.A. Endothelial cells, macrophages, neutrophils and neuronal cells can generate 02- and -NO (endothelial-derived relaxation factor) at increased rates when activated or metabolically stressed. Reaction of 0 2 and .NO yields peroxynitrite anion (ONOO-), a potent oxidant which will oxidize both non-protein and protein sulfhydryls. At pH 7.4 and 37oC, apparent second order rate constants were 5900 M-l.s-1 and 1700 M I . s I for the reaction of peroxynitrite anion with free cysteine and the single thiol of albumin, respectively. These rate constants are three orders of magnitude greater than the corresponding rate constants for reaction of hydrogen peroxide with these sulfhydryls (pH 7.4). Unlike H202, which oxidizes thiolate anion, peroxynitrite anion reacted preferentially with the undissociated form of the thiol group. Peroxynitrite-dependent cysteine oxidation was 90% cyanide and DTT reducible, indicating that cystine was the main oxidation product. Electrophoretic analysis revealed that peroxynitrite did not result in disulfide-mediated dimerization or peptide cleavage of BSA at pH 7.4. Peroxynitrite oxidized the BSA-thiol group to an arsenite non-reducible product, while borohydride recovered 50% of the BSA-thiol, suggesting oxidation beyond sulfenic acid. Peroxynitrous acid was a less effective thiol oxidizing agent than its anion, and presumably the oxidation was mediated by its apparent decomposition products, i.e. hydroxyl radical and nitrogen dioxide. These observations show that secondary reactions in the vascular space between 02- and -NO can exert cytotoxic effects mediated, in part, by alteration of tissue sulfhydryl homeostasis.
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CYTOCHROME b 5 MEDIATED REDOX CYCLING OF ESTROGENS. Deodutta Roy, Henry W. Strobel 1, and Joachim G. Liehr. Dept. of Pharmacology & Toxicoio~gy, Univ. of Texas Med. Branch, Galveston, TX 77550 and Dept. of Biochemistry & MoL Biology, Univ. of Texas Health Sci. Ctr., Houston, TX 77225, USA.
Previously we have demonstrated microsomai cytochrome P 450catalyzed redox cycling of estrogens. In this study, we investigated the role of cytochrome b 5 ('o5) in redox cycling. Pure P 450c and peroxides oxidized diethylstilbestrol (DES) to diethylstilbestrol-4',4"-qulnone (DES Q). This oxidation by H20 2 was doubled by addition of b 5 to P 450c, but did not proceed with b 5 alone. The stimulation by b 5 of the P 450c-catalyzed oxidation of DES to DES O occurred via modulation of the Vmax of P 450c rather than of the Kin. DES O was reduced to DES by purified bs and NADH-dependent b5 reductase. Pretreatment of micrusomes with an antibody of b 5 reductase inhibited microsomal NADH-dependent reduction of DES O to DES. The b 5 likely participates in the oxidation of DES to DES O by interacting with P-450c and in the reduction of DES O to DES by interacting with b5 reductase. Thus, the study demonstrates that b5 plays an active role in biological oxidation and reduction reactions. Supported by NIH, NCI (CA 43232, CA 43233).
EFFECT~S OF OXYGEN FREE RADICALS ON HUMAN ERYTHR~: PROTECrlVE EFFECTS OF DEFEROXAMINE AND ALI/3PURINOL. Moheb A. Rashid, Olof Jonsson, Donald G.Roberts, and C45ran William-Olsson. Dept. of Thoracic&Cardiovascular Surgery,Sahlgrenska Hospital,413 45 Gothenburg,Sweden.
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Oxygen free radicals(OFR) are produced by blood trauma during cardiopulmonary bypass(CPB) in cardiac surgery leading to reduced blood cell theology and disturbed microcirculation.Deferoxamine(DFX) an iron chelator and allopurinol (ALLP) an antioxidant are OFR scavengers. Their protective effects on RFR induced red blood cell (RBC) damage in the presence of mechanical blood trauma was compared by estimating RBC filtrability(RFR) during 4 hours in 12 experiments using fresh human RBCs.Each exp. had 6 groups:-1: control; 2: hypoxa nthine (HX )&xa nthine oxida se (XOD ) 3:10 mic.g.DFX;4:10 mic.g.AI/~;5:HX&XOD+10 mic.g. DFX; 6 :HX&XOD+I 0 mic. g .ALLP. There was significant improvement of RFR in groups 3,4,5 and 6 compared to groups I&2(p<0.05).DFX showed significantly better RFR(42.4+5&40.0+1 mic.L/sec) than ALLP (24.7+2&22.2+4 mic_L/sec)Tp=0.00003&p=0.004 ) at l&2 h~urs respectively. In conclusion,both DFX and ALLP protected RBCs:DFX appears to enhance membrane stability and improve RFR,probably by neutralizing intracellular OFR. This should lead to further studies in cardiac surgical patients undergoing CPB.
SUBSTRATE INHIBITION OF XANTHINE OXlDASE AND ITS RELATIONSHIP WITH SUPEROXlDE RADICAL PRODUCTION. Homero Rubbo, Rafael Radi and Eugenio Prodanov. Departamento de Bioquimica. Facultad de Medicina, Universidad de la Republica. Montevideo, Uruguay. A possible relationship between intramolecular electron transport and substrate inhibition was studied in reactions catalyzed by xanthine oxidase. Using hypoxanthine and xanthine as substrates, the kinetics of product formation (uric acid and superoxide anion radical) were followed spectrophotometrically. Experiments with hypoxanthine showed an inhibition which fits an equation of the classical form: v = V.S/(K m +a.S + S2/Ki), with reaction velocity decreasing with increasing substrate concentration, from 1.0 to 9.3 x 10-4 M. With xanthine, the type of inhibition was more complex, perhaps because inactivation only occurs at very high xanthine concentrations. With both substrates, inhibition was not reverted by addition of SOD and/or catalase. The percent of monovalent electron flux to oxygen diminished by increasing substrate concentration in the range of 0.5 to 6.0 x 10-5 M xanthine or hypoxanthine. Over this range of substrate concentrations, increasing substrate concentration increased the percent of monovalent flux and at the same time diminishing the velocity of uric acid production. This effect was pH-dependent, more significant at pH 9.5 than pH 8.3, and more so for hypoxanthine than with xanthine. It is concluded that the alteration of the internal electron transport by high substrate concentrations favors the monovalent pathway and interferes with enzyme catalytic activity.
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