- Email: [email protected]
Hyperoxia inactivates aconitase and reduces catalase levels in the lung of CuZnSOD transgenic mice
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i
MITOCHONDRIAL -OXIDANT INTERACTIONS Bruce S. Kristal,19zAlison J. Moskowitz,s M&hail B. Bogdanov.4 M. Flint Bealz, Karen E. Vigneau-Callahan,5 Wayne R. Matson5 ‘Burke Medical Research Institute; 2Comell Univ. Medical College; 3Comell Univ.; 4Mass. General Hospital; sESA, Inc. Inappropriate univalent reduction of oxygen by the mitochondrial electron transport chain is a major endogenous source of reactive species, especially under pathological conditions. Reciprocally, mitochondria are susceptible to oxidant-mediated damage. We have utilized two HPLC-based methods to further understand the interactions between generation of oxidants, mitochondrial defense systems, and mitochondrial dysfunction: One method enables simultaneous analysis of as many as 600 redox active compounds from mitochondria; the other enables quantitation of 8-hydroxydeoxyguanosine (8.OHdG) at sub-pg/ml levels. Results from four studies in rat liver mitochondria show: 1) Methodology: Use of Hepes buffers alters concentrations of some redox active compounds in the mitochondrial fraction 2) Respiration: Minimal or no changes in mitochondrial redox-active compounds are observed during State 3 or State 4 respiration, but exposure to mitochondrial poisons results in changes in specific redox-active species. 3) DNA damage: 8-OH dG is generated and released by mitochondria during long incubations with succinate or glutamate/malate, but not with ascorbateTTMPD,implicating radical generation at Center P of the respiratory chain in mtDNA damage. 4) Permeability transition (PT): PT induction is preceded and accompanied by sequential depletion of mitochondrial antioxidants and the appearance of novel redox-active species; these changes are qualitatively and quantitatively altered by t-butylhydroperoxide. In summanL,,technologicaladvances have enabled more detailed probes of rmtochondrial-oxidant interactions.
HYPEROXIA INACTIVATES ACONITASE AND REDUCES CATALASE LEVELS IN THE LUNG OF CUZNSOD TRANSGENIC MICE
expression.
I 72
/
43210
Glutathione (GSH) plays an important role in cellular antioxidation and detoxification of reactive oxygen species (ROS) and xenobiotics. Thus maximizing GSH concentrations in selected target tissues may protect against ROS-mediated tissue damage. Although cysteine is the limiting amino acid for GSH synthesis, it is rapidly oxidized and can be toxic. The objective of this study is to compare the efficacy of 4 different cysteine prodrugs as dietary supplements to enhance tissue GSH levels under different nutritional status. CD-1 mice were randomized into 4 groups fed a 05% (deficient), 75% (low), 15% (normal) or 30% (high) protein diet. Following 2 week feeding, each group was further divided into 5 subgroups: a control and 4 groups supplemented with N-a-1 cysteine (NAC), L-2+m-4-thiazolidine-carboxylic acid (OTC), methionine (MET) or GSH. After 1 week of supplementation, lung, liver, heart, colon and brain were removed and analyzed for total GSH status. Mice fed a protein 0.5% showed markedly lower total GSH levels in several tissues, in&ding spleen, lung, colon and liver. All 4 cysteine pmdrugs increased tissue GSH in the protein deficient mice to the level observed in mice fed 15% protein. However, supplementation of cysteine prodrugs did not enhance tissue GSH level in mice fed a normal or high protein diet. For the 0.5% protein group, dietary supplementation of GSH is most effective in increasing tissue GSH levels in liver, lung and colon while NAC and MET were able to selectively increase spleen GSH level. All 4 prodrugs were equally effective in enhancing total GSH in the heart.) Finding tissuespecificity of different GSH precurso rs will provide information to develop strategies to protect target tissues against ROS-mediated damage.
s34
Yu-Hd Columbus,
Tyli, Andrew Razum OH, and Cepbdon,
and Tammy
M. Br
Inc.WestChestn. P 2
Tke Ohio St&
Oxidative stress and cellular redox status modulate the activity of aconitase, a redox-sensitive iron-sulfur (Fe-S) protein.t Reactive oxygen species (ROS), in particular 02-L can reversibly inactivate aconitase, first by oxidation of the Fe-S cluster followed by loss of Fe(II) from the c1uster.t This results in increak in intracellular ‘free’ iron and may therefore potentiate the production of other ROS through Fenton based chemistry. The objective of this study was to determine if hyperoxiacBR>exp&ure and variable CuZnSOD expression. an exogenous and endogenous oxklative stress, respectively, would modulate aconitase activity and trace mineral levels in lung and liver tissue. Transgenic mice with 4 different levels of CuZnSOD expression, overexpresser (+++I, normal (++), hemizygous (+/-) and homozygous c-/-j, were exposed to either hypemxia (85202) or normoxia (219002) for 5 days. In the lung, aconitase activity was significantly reduced following exposure to hypemxia but was not affected by CuZnSOD expression. Conversely in the liver, aconitase was not affected by hyperoxia, but was reduced by half in the SOD(-/-) mice compared to all other genotypes. Similar to aconitase, hyperoxia reduced catalase expression by 50% in the lung but had no effect on liver catalase. We also examined lung and liver tissue levels of the pro-oxidant metals Fe and Cu as well as the antioxidant metal Zn in order to determine if the tissue concentrations of these minerals are affected by variations in CuZnSODgene expression and/or oxidative stress. Total Fe and Zn
levels in the lung were reduced, but ceruloplasmin, a Cu-cuntainlng protein was increased after hyperoxia. Total liver Fe was inversely correlated, but total Cu was directly correlated to CuZnSOD
I 73 I
THE EFFICACY OF CYSTEINE PRODRUGS IN SELEmIVE ENHANCEMENT OF TISSUE GLUTATHIONE (GSH) IN CD-l MICE u Gary Stoner, nnd Tammy Bray, Tke OhioSt& University, Columbus, OH,
Mark Lmy, Unionsity,
OXYGEN
1
POLY(ADP-RIEIOSE) POLYMERASE, OXlDATIVE STRESS AND SIGNAL TRANSDUCTION IN ATAXIA TELANGIECTASIA John C. Mare&i, Sonia Flores, Gisela Viatitis and Joe McCord, Webb- Waring Institute, Denver, Colorado In a&a-telangiectasia (A-T), there is evidence for oxidative stress as a consequence of the aberrant expression of the protein that is responsible for the disorder (ATM). A-T cells have chronically activated poly(ADP-ribose) polymerase (PARP), aDNA repair enzyme which causes a depletion of cellular levels of NAD’ and ATP via the production of poly(ADP-ribose). This, coupled with the inability to survey and repair damage to DNA results in an energetically and oxidatively imbalanced cell. In fibroblasts from AT patients, we found an increase in the steady-state level of PARP activity and a decrease in cellular NAD’. When treated with PARP inhibitorsO-aminobenzamideor 1,5-dihydroisoquinoline)thesecells showed a dose-dependant increase in growth rates. These experiments demonstrate that A-T fibroblaats have altered PARP activity and NAD’ content providing evidence for a state of oxidative stress. When the nuclei were assayed for transcription factors, A-T ceils showed a dramatic decrease in the steady-state levels of NF-ti, in contrast to what has been reported by others for a transformed A-T cell line. Reports indicate that wild-type ATM phosphorylates I-KB, therefore we propose that the mutations in ATM disrupt the normal NF-KB signal transduction pathway rendering the cell unable to respond to oxidative stress. These factors may contribute to the radiation sensitivity and apoptosis in A-T.
’ 9
8
i
MITOCHONDRIAL -OXIDANT INTERACTIONS Bruce S. Kristal,19zAlison J. Moskowitz,s M&hail B. Bogdanov.4 M. Flint Bealz, Karen E. Vigneau-Callahan,5 Wayne R. Matson5 ‘Burke Medical Research Institute; 2Comell Univ. Medical College; 3Comell Univ.; 4Mass. General Hospital; sESA, Inc. Inappropriate univalent reduction of oxygen by the mitochondrial electron transport chain is a major endogenous source of reactive species, especially under pathological conditions. Reciprocally, mitochondria are susceptible to oxidant-mediated damage. We have utilized two HPLC-based methods to further understand the interactions between generation of oxidants, mitochondrial defense systems, and mitochondrial dysfunction: One method enables simultaneous analysis of as many as 600 redox active compounds from mitochondria; the other enables quantitation of 8-hydroxydeoxyguanosine (8.OHdG) at sub-pg/ml levels. Results from four studies in rat liver mitochondria show: 1) Methodology: Use of Hepes buffers alters concentrations of some redox active compounds in the mitochondrial fraction 2) Respiration: Minimal or no changes in mitochondrial redox-active compounds are observed during State 3 or State 4 respiration, but exposure to mitochondrial poisons results in changes in specific redox-active species. 3) DNA damage: 8-OH dG is generated and released by mitochondria during long incubations with succinate or glutamate/malate, but not with ascorbateTTMPD,implicating radical generation at Center P of the respiratory chain in mtDNA damage. 4) Permeability transition (PT): PT induction is preceded and accompanied by sequential depletion of mitochondrial antioxidants and the appearance of novel redox-active species; these changes are qualitatively and quantitatively altered by t-butylhydroperoxide. In summanL,,technologicaladvances have enabled more detailed probes of rmtochondrial-oxidant interactions.
HYPEROXIA INACTIVATES ACONITASE AND REDUCES CATALASE LEVELS IN THE LUNG OF CUZNSOD TRANSGENIC MICE
expression.
I 72
/
43210
Glutathione (GSH) plays an important role in cellular antioxidation and detoxification of reactive oxygen species (ROS) and xenobiotics. Thus maximizing GSH concentrations in selected target tissues may protect against ROS-mediated tissue damage. Although cysteine is the limiting amino acid for GSH synthesis, it is rapidly oxidized and can be toxic. The objective of this study is to compare the efficacy of 4 different cysteine prodrugs as dietary supplements to enhance tissue GSH levels under different nutritional status. CD-1 mice were randomized into 4 groups fed a 05% (deficient), 75% (low), 15% (normal) or 30% (high) protein diet. Following 2 week feeding, each group was further divided into 5 subgroups: a control and 4 groups supplemented with N-a-1 cysteine (NAC), L-2+m-4-thiazolidine-carboxylic acid (OTC), methionine (MET) or GSH. After 1 week of supplementation, lung, liver, heart, colon and brain were removed and analyzed for total GSH status. Mice fed a protein 0.5% showed markedly lower total GSH levels in several tissues, in&ding spleen, lung, colon and liver. All 4 cysteine pmdrugs increased tissue GSH in the protein deficient mice to the level observed in mice fed 15% protein. However, supplementation of cysteine prodrugs did not enhance tissue GSH level in mice fed a normal or high protein diet. For the 0.5% protein group, dietary supplementation of GSH is most effective in increasing tissue GSH levels in liver, lung and colon while NAC and MET were able to selectively increase spleen GSH level. All 4 prodrugs were equally effective in enhancing total GSH in the heart.) Finding tissuespecificity of different GSH precurso rs will provide information to develop strategies to protect target tissues against ROS-mediated damage.
s34
Yu-Hd Columbus,
Tyli, Andrew Razum OH, and Cepbdon,
and Tammy
M. Br
Inc.WestChestn. P 2
Tke Ohio St&
Oxidative stress and cellular redox status modulate the activity of aconitase, a redox-sensitive iron-sulfur (Fe-S) protein.t Reactive oxygen species (ROS), in particular 02-L can reversibly inactivate aconitase, first by oxidation of the Fe-S cluster followed by loss of Fe(II) from the c1uster.t This results in increak in intracellular ‘free’ iron and may therefore potentiate the production of other ROS through Fenton based chemistry. The objective of this study was to determine if hyperoxiacBR>exp&ure and variable CuZnSOD expression. an exogenous and endogenous oxklative stress, respectively, would modulate aconitase activity and trace mineral levels in lung and liver tissue. Transgenic mice with 4 different levels of CuZnSOD expression, overexpresser (+++I, normal (++), hemizygous (+/-) and homozygous c-/-j, were exposed to either hypemxia (85202) or normoxia (219002) for 5 days. In the lung, aconitase activity was significantly reduced following exposure to hypemxia but was not affected by CuZnSOD expression. Conversely in the liver, aconitase was not affected by hyperoxia, but was reduced by half in the SOD(-/-) mice compared to all other genotypes. Similar to aconitase, hyperoxia reduced catalase expression by 50% in the lung but had no effect on liver catalase. We also examined lung and liver tissue levels of the pro-oxidant metals Fe and Cu as well as the antioxidant metal Zn in order to determine if the tissue concentrations of these minerals are affected by variations in CuZnSODgene expression and/or oxidative stress. Total Fe and Zn
levels in the lung were reduced, but ceruloplasmin, a Cu-cuntainlng protein was increased after hyperoxia. Total liver Fe was inversely correlated, but total Cu was directly correlated to CuZnSOD
I 73 I
THE EFFICACY OF CYSTEINE PRODRUGS IN SELEmIVE ENHANCEMENT OF TISSUE GLUTATHIONE (GSH) IN CD-l MICE u Gary Stoner, nnd Tammy Bray, Tke OhioSt& University, Columbus, OH,
Mark Lmy, Unionsity,
OXYGEN
1
POLY(ADP-RIEIOSE) POLYMERASE, OXlDATIVE STRESS AND SIGNAL TRANSDUCTION IN ATAXIA TELANGIECTASIA John C. Mare&i, Sonia Flores, Gisela Viatitis and Joe McCord, Webb- Waring Institute, Denver, Colorado In a&a-telangiectasia (A-T), there is evidence for oxidative stress as a consequence of the aberrant expression of the protein that is responsible for the disorder (ATM). A-T cells have chronically activated poly(ADP-ribose) polymerase (PARP), aDNA repair enzyme which causes a depletion of cellular levels of NAD’ and ATP via the production of poly(ADP-ribose). This, coupled with the inability to survey and repair damage to DNA results in an energetically and oxidatively imbalanced cell. In fibroblasts from AT patients, we found an increase in the steady-state level of PARP activity and a decrease in cellular NAD’. When treated with PARP inhibitorsO-aminobenzamideor 1,5-dihydroisoquinoline)thesecells showed a dose-dependant increase in growth rates. These experiments demonstrate that A-T fibroblaats have altered PARP activity and NAD’ content providing evidence for a state of oxidative stress. When the nuclei were assayed for transcription factors, A-T ceils showed a dramatic decrease in the steady-state levels of NF-ti, in contrast to what has been reported by others for a transformed A-T cell line. Reports indicate that wild-type ATM phosphorylates I-KB, therefore we propose that the mutations in ATM disrupt the normal NF-KB signal transduction pathway rendering the cell unable to respond to oxidative stress. These factors may contribute to the radiation sensitivity and apoptosis in A-T.
’ 9
8