- Email: [email protected]
The role of iron in the cytotoxicity of crocidolite
520
7:17
Session 7: Oxidative Mechanisms of Cell “Death” FORMATION OF HYDROXYL RADICALS ON REDUCTION OF HYPOCHLOROUS ACID Luis P. Candeias; Michael R. L. Stratford and Peter Wardman Cancer Research Campaign Gray Laboratory PO Box 100, Mount Vernon Hospital, Northwood, Middlesex. HA6 2JR, UK Hypochlorous acid (HOCI) is an oxidant released by activated neutrophils that has been suggested to contribute to the bactericidal, tumouricidal and inflammatory effects of these cells. Although HOCI is known to react with many biological compounds and anti-inflammatory drugs, the reaction mechanisms have been established in only a few cases. In particular, the suggested involvement of free radical intermediates deserves fuher investigation. In this work we show that HOCI reacts with reducing radicals (ea -, CO,‘- and O,‘-) to yield hydroxyl radicals (‘OH). Similar resu9 ts were obtained with reduced metal ions (Fe(ll) and Cu(l)). Taurine (2-aminoethanesulphonic acid) and glutathione react with HOCI without formation of OH. These results show the dual behaviour of HOC1 as either one-electron oxidant (source of free radicals) or electrophilic (chlorinating) species. The biological consequences of the superoxide anion (Os’-) generated by activated neutrophils are still debated. The reaction with HOC1 with formation of hydroxyl radicals demonstrated in the present work is a possible mechanism of biological damage by phagocytic cells. l
7:19
PYRIDINE NUCLEOTIDES AND THE REDUCTION OF GSSG IN HYPGXIC HEART TISSUE EXPOSED TO DIAMlDE James P. Kehrer, Loren G. Lund and Thomas Paraidathathu Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, TX, 788712 USA A tissue’s ability to maintain normal redox balance under an oxidative or reductive stress is believed to be energy-dependent. However, while the production of energy and reducing equivalents are closely linked, they are separate biochemical events. The supply of reducing equivalents, not energy, determines the ability of a tissue to defend against oxidative injury. The current study utilized HPLC analyses of intact heart tissue perfused by the Langendorff technique under hypoxic or oxygenated conditions with medium containing 2.5 mM calcium. The infusion of 800 l_tM diamide for 5 min after 60 min of normoxia or hypoxia oxidized 70-80% of cardiac glutathione (GSH). Subsequently, 82% (nonnoxia) or 72% (hypoxia) of the glut&none dlsulflde (GSSG) formed by diamide was reduced after a 25 mm recovery period. Almost identical results were obtained when the experiment was repeated in the absence of 10 mM glucose. Cardiac ATP and phosphocreatine levels were depleted greater than 99% by hypoxia in the absence of glucose. NADH increased during hypoxia while NADPH was unchanged. The infusion of diamide during hypoxia (without glucose) decreased NADH from 0.65 to 0.48 nmol/mg dry weight. This was still above normoxic levels of 0.2. There was a 15% increase in NADH during the hypoxic recovery period indicating a continuing source of reducing equivalents was available. Only 36% and 3% of the GSSG formed by diamide was reduced after the 25 min recovery period in hearts pretreated for 30 mm with 100 or 200 pM BCNU (which inhibited glutathione reductase activity by 74 and 85%. respectively). This demonstrated the requitement for NADPH. Replacing glucose with 2-deoxyglucose (5 mM) to inhibit glycolysis failed to prevent the reduction of GSSG after diamide and had little effect on NADPH or NADH levels. These data show that intact hypoxic heart tissue is able to respond to the oxidant dinmide as well as normoxic tissue and that NADPH is protected at the expense of NADH. Reducing equivalents appear to come from pathways unrelated to glycolysis. These results are in contrast to work in hepatocytes indicating significant differences between organs or perhaps isolated cells versus an intact tissue.
THE ROLE OF CROCIDOLITE C. Chao, R. Department
IRON
IN
THE
CYTOTOXICITY
OF
7:18
Lund and A. Aust, Shea, L of Chem. and Biochem., Utah State Univ., Logan, UT 84322-0300 USA.
Asbestos catalyzes the same oxidative reactions that iron does, i.e. ,OH formation, lipid peroxidation, and DNA oxidation. We propose that iron is responsible for the carcinogenicity of asbestos. The fibers can have intrinsic iron or can acquire iron on the surface. To examine the role of iron in the cytotoxicity of asbestos, human lung carcinoma cells (A549) were treated with crocidolite in 3 types,of media, F-12 (3 PM Fe), DMEM (0.25 pm Fe), and IMDM (0 /AM Fe or 3 pm). At 2 pg/cm2 crocidolite the survival was 9%, 20%, 26% and 8%, respectively. To determine the fate of 5SFe mobilized intracellularly from neutron activated crocidolite, a LMW fraction < 10,000 Fe MW) was compared with total s\ mobilized (> 10,000 x g supernatant). The LMW '5Fe increased with the treatment dose and showed an inverse linear relationship with cytotoxicity. These results suggest that acquired or intrinsic iron associated with asbestos may be responsible for its cytotoxicity and that mobilization into a LMW fraction may play an important role. (Supported by NIH, ES05782 & ES05814)
DEATHOFPOLYMORPHONUCLEARLEUKOCYTES AFTERTHERESPIRATORYBURSTBYOXYGENRADICALS. Yashige Kotake, Mari Tanigawa, and TON Tanigawa Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, U.S.A. Superoxide has been suggested to be a cause of death of polymorphonuclear leukocytes (PMNs) after the respiratory burst. In order to obtain suppoiting evidence of this notion the survival rate of human PMNs in vitro after the respiratory burst was measured in the Presence of various agents which affect their capacity of superoxide generation. The survival rate determined from the cell count of PMNs inHBSS buffer abruptly decreased 80 min after aerobic incubation at 37oC with a respiratory burst stimulant, phorbol myristate acetate (PMA). In 95 min after the stimulation, the survival rate fell to 50 W. However, substantial increase in the survival time was obtained when PMA-stimulated PMNs were preincubated before stimulation in the presence of an NADPH ox&se inhibitor or a protein kinase C inhibitor. For example, diphenylene iodonium, a potent NADPH ox&se inhibitor extended 50 %-survival time to 280 min. These inhibitors are capable of suppressing superoxide generation, suggesting that the cell death is mediated by superoxide.. Loss of ATP by the respiratory burst was shown not to be a cause of the cell death because preincubation of PMNs with an ATP depleting agent did not decrease the survival rate. Presence of a PMNproteinase inhibitor showed minimal effect on the survival rate, indicating that the proteinase which may be activated by the respiratory burst is not a cause of cell death. Consistent decrease of PMNs'superoxide generation monitored with cytochrome. c reduction was obtained in the presence of agents used in the survival rate measurement.
7:20
7:17
Session 7: Oxidative Mechanisms of Cell “Death” FORMATION OF HYDROXYL RADICALS ON REDUCTION OF HYPOCHLOROUS ACID Luis P. Candeias; Michael R. L. Stratford and Peter Wardman Cancer Research Campaign Gray Laboratory PO Box 100, Mount Vernon Hospital, Northwood, Middlesex. HA6 2JR, UK Hypochlorous acid (HOCI) is an oxidant released by activated neutrophils that has been suggested to contribute to the bactericidal, tumouricidal and inflammatory effects of these cells. Although HOCI is known to react with many biological compounds and anti-inflammatory drugs, the reaction mechanisms have been established in only a few cases. In particular, the suggested involvement of free radical intermediates deserves fuher investigation. In this work we show that HOCI reacts with reducing radicals (ea -, CO,‘- and O,‘-) to yield hydroxyl radicals (‘OH). Similar resu9 ts were obtained with reduced metal ions (Fe(ll) and Cu(l)). Taurine (2-aminoethanesulphonic acid) and glutathione react with HOCI without formation of OH. These results show the dual behaviour of HOC1 as either one-electron oxidant (source of free radicals) or electrophilic (chlorinating) species. The biological consequences of the superoxide anion (Os’-) generated by activated neutrophils are still debated. The reaction with HOC1 with formation of hydroxyl radicals demonstrated in the present work is a possible mechanism of biological damage by phagocytic cells. l
7:19
PYRIDINE NUCLEOTIDES AND THE REDUCTION OF GSSG IN HYPGXIC HEART TISSUE EXPOSED TO DIAMlDE James P. Kehrer, Loren G. Lund and Thomas Paraidathathu Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, TX, 788712 USA A tissue’s ability to maintain normal redox balance under an oxidative or reductive stress is believed to be energy-dependent. However, while the production of energy and reducing equivalents are closely linked, they are separate biochemical events. The supply of reducing equivalents, not energy, determines the ability of a tissue to defend against oxidative injury. The current study utilized HPLC analyses of intact heart tissue perfused by the Langendorff technique under hypoxic or oxygenated conditions with medium containing 2.5 mM calcium. The infusion of 800 l_tM diamide for 5 min after 60 min of normoxia or hypoxia oxidized 70-80% of cardiac glutathione (GSH). Subsequently, 82% (nonnoxia) or 72% (hypoxia) of the glut&none dlsulflde (GSSG) formed by diamide was reduced after a 25 mm recovery period. Almost identical results were obtained when the experiment was repeated in the absence of 10 mM glucose. Cardiac ATP and phosphocreatine levels were depleted greater than 99% by hypoxia in the absence of glucose. NADH increased during hypoxia while NADPH was unchanged. The infusion of diamide during hypoxia (without glucose) decreased NADH from 0.65 to 0.48 nmol/mg dry weight. This was still above normoxic levels of 0.2. There was a 15% increase in NADH during the hypoxic recovery period indicating a continuing source of reducing equivalents was available. Only 36% and 3% of the GSSG formed by diamide was reduced after the 25 min recovery period in hearts pretreated for 30 mm with 100 or 200 pM BCNU (which inhibited glutathione reductase activity by 74 and 85%. respectively). This demonstrated the requitement for NADPH. Replacing glucose with 2-deoxyglucose (5 mM) to inhibit glycolysis failed to prevent the reduction of GSSG after diamide and had little effect on NADPH or NADH levels. These data show that intact hypoxic heart tissue is able to respond to the oxidant dinmide as well as normoxic tissue and that NADPH is protected at the expense of NADH. Reducing equivalents appear to come from pathways unrelated to glycolysis. These results are in contrast to work in hepatocytes indicating significant differences between organs or perhaps isolated cells versus an intact tissue.
THE ROLE OF CROCIDOLITE C. Chao, R. Department
IRON
IN
THE
CYTOTOXICITY
OF
7:18
Lund and A. Aust, Shea, L of Chem. and Biochem., Utah State Univ., Logan, UT 84322-0300 USA.
Asbestos catalyzes the same oxidative reactions that iron does, i.e. ,OH formation, lipid peroxidation, and DNA oxidation. We propose that iron is responsible for the carcinogenicity of asbestos. The fibers can have intrinsic iron or can acquire iron on the surface. To examine the role of iron in the cytotoxicity of asbestos, human lung carcinoma cells (A549) were treated with crocidolite in 3 types,of media, F-12 (3 PM Fe), DMEM (0.25 pm Fe), and IMDM (0 /AM Fe or 3 pm). At 2 pg/cm2 crocidolite the survival was 9%, 20%, 26% and 8%, respectively. To determine the fate of 5SFe mobilized intracellularly from neutron activated crocidolite, a LMW fraction < 10,000 Fe MW) was compared with total s\ mobilized (> 10,000 x g supernatant). The LMW '5Fe increased with the treatment dose and showed an inverse linear relationship with cytotoxicity. These results suggest that acquired or intrinsic iron associated with asbestos may be responsible for its cytotoxicity and that mobilization into a LMW fraction may play an important role. (Supported by NIH, ES05782 & ES05814)
DEATHOFPOLYMORPHONUCLEARLEUKOCYTES AFTERTHERESPIRATORYBURSTBYOXYGENRADICALS. Yashige Kotake, Mari Tanigawa, and TON Tanigawa Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, U.S.A. Superoxide has been suggested to be a cause of death of polymorphonuclear leukocytes (PMNs) after the respiratory burst. In order to obtain suppoiting evidence of this notion the survival rate of human PMNs in vitro after the respiratory burst was measured in the Presence of various agents which affect their capacity of superoxide generation. The survival rate determined from the cell count of PMNs inHBSS buffer abruptly decreased 80 min after aerobic incubation at 37oC with a respiratory burst stimulant, phorbol myristate acetate (PMA). In 95 min after the stimulation, the survival rate fell to 50 W. However, substantial increase in the survival time was obtained when PMA-stimulated PMNs were preincubated before stimulation in the presence of an NADPH ox&se inhibitor or a protein kinase C inhibitor. For example, diphenylene iodonium, a potent NADPH ox&se inhibitor extended 50 %-survival time to 280 min. These inhibitors are capable of suppressing superoxide generation, suggesting that the cell death is mediated by superoxide.. Loss of ATP by the respiratory burst was shown not to be a cause of the cell death because preincubation of PMNs with an ATP depleting agent did not decrease the survival rate. Presence of a PMNproteinase inhibitor showed minimal effect on the survival rate, indicating that the proteinase which may be activated by the respiratory burst is not a cause of cell death. Consistent decrease of PMNs'superoxide generation monitored with cytochrome. c reduction was obtained in the presence of agents used in the survival rate measurement.
7:20