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Oxygen radical formation by epiphyseal chondrocytes causes changes in cartilage collagen synthesis
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Oxidative Stress Genes and Proteins O X Y G E N R A D I C A L F O R M A T I O N BY E P I P H Y S E A L CHONDROCYTES CAUSES C H A N G E S IN C A R T I L A G E COLLAGEN SYNTHESIS Irving M. Shapiro, Toshikazu Tokuoka, Elizabeth Forbes, Kristine DeBolt and Manrizio Pacifici Departments of Biochemistry and Anatomy-Histology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, U.S.A. The epiphyseal growth plate is the site for the growth and elongation of bone. Our previous studies of this tissue indicate that reactive oxygen species (ROS) may be generated by cells in the epiphyseal hypertrophic region. To ascertain how ROS generation affects the synthesis of the extraeellular matrix proteins we set up a cell culture system to mimic events that occur in the development of the growth plate. Chondrocytes were isolated from the growth plates of chick embryo tibiae by a collagenase digestion procedure. The cells were cultured in a medium supplemented with ascorbate in the presence and absence of Fe(II). Measurements of thiobarbituric acid reactive substance (TBARS) production by these cultured chondroeytes showed that both ascorbate and Fe(II) activated ROS-dependent lipid peroxidation. Stimulation of ROS formation had a profound effect on protein synthesis. When the cells were labeled with [35]S-methionine for a short time period and the newly synthesized proteins were separated by PAGE in a 6% gel, changes were seen in a number of identified proteins; the most significant effect was an increase in the mobility of pro alpha Type II and Type X collagens. For both of these collagens, the lowered molecular weight was probably due to protein under-hydroxylation. It was concluded that ROS generation by cells of the epiphyseal growth cartilage can modify the chemical nature of the extracellular matrix by regulating the synthesis of proteins that are required for normal tissue development and mineralization.
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YEAST AS A MODEL FOR OXIDANT INJURY. MECHANISM OF OXYGEN REGULATION OF ELECTRON TRANSPORT ACTIVITY Richard M. Wright, Webb-Waring Lung Institute at University of Colorado HSC,Denver, Colorado 80262 We are using the yeast Saccharomyces cerevisiae to model mechanisms of oxidant injury. The mitochondrial electron transport chain is potentially a major source of intracellular oxygen radicals, and its activity is strictly coupled to oxygen level. As oxygen tension falls activity of specific electron transport components falls. As a first step in our analyses, we have examined how oxygen regulates these changes inelectron transport activity. We find that hypoxic cultures are a) depressed in electron transport activity, b) show loss of the protein components at cytochrome aa3 (and elsewhere), e) have greatly decreased levels of steady state mRNAs for cytochrome aa3 genes, d) show decreased transcription of cytochrome aa3-1ac Z fusion plasmids, and e) the site conferring regulation by oxygen is the same as that conferring heme regulation of eytochrome aa3 genes. Our results suggest that the oxygen sensing mechanism for cytochrome aa3 is transduced through a heme dependent pathway as it is for cytochrome c. Interestingly, under conditions of hyperoxia we do not observe the corresponding effect at the level of transcription of cytoehrome aa3 genes or the cytochrome c gene. Perhaps in yeast hyperoxie injury is exerted entirely at the level of enzyme kinetics or activity and not at the level of gene expression.
IN SITU ANALYSIS OF MANGANESE SUPEROXIDE DISMUTASE (MnSOD) IN ALVEOLAR TYPE II CELLS OF HYPEROXIC RATS. Renaud Vincent, Ye-Shih Ho, Ling-Yi Chang, Jan W. Slot, and James D. Crapo. Duke University Medical Center, Durham, NC, 27710, and University of Utrecht, Utrecht, The Netherlands.
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Rats exposed to 85% 0 2 for 5-7 days develop a tolerance to hyperoxia, which is manifested by prolonged survival in 100% O~, a treatment normally lethal within 60 hours. This tolerance m attributed to a selective increase of antioxidant defenses in the lungs, compensating for an higher intracellular flux of activated oxygen species. Total lung MnSOD is particularly increased following prolonged exposure to 85% 0 2. We have studied in situ by immunocytochemistry the time-cours~ and the magnitudeoft-~ee changes of MnSOD in alveolar type II cells, a probable locus of the antioxidant response. Rats were exposed to 85% Oafor 7 days and 14 days. The lungs were fixed, embedded in LP.-White, and sections for EM were labeled with anti-rat MnSOD antiserum followed by protein A-gold. Induction of MnSOD after 7 days was confirmed by the observation of llbroblasts and type II cells with high levels of the protein in their mitochondria. However, the response was not uniform. In average, the labeling density (gold/urn2) over mitochondria of type II cells increased by 22% above control value after 7 days, and 66% (p<0.05) after 14 days. The data support the concept of an increased concentration of MnSOD in the mitochondria, but the estimated contribution of type II cells to the total lung MnSOD response is mainly through hypertrophic and hyperplastic changes, and by an increased mitochondrial volume at 14 days (total type II cells MnSOD at 7 days, + 155% above controls; 14 days, + 413%). The steady-state level of mRNA for MnSOD in total lungs was found to increase rapidly during exposure to 85% O 2 (3 days) but returned to baseline by 14 days. These results contrasted with the time-course of enzyme increase, which appeared as a delayed event. We suggest that an early response of MnSOD in the lungs may be partly driven by inflammatory events, while the enzymatic changes measured at 14 days and the accumulation of mitochondria may constitute a direct oxygen effect, reflecting oxygen adaptation.
ADAPTIVE RESPONSE TO HYDROGEN PEROXIDE IN MAMMALIAN CELLS Anne G. Wiese, Robert E. Paciflci, and Kelvin J. A. Davies Institute for Toxicology & Department of Biochemistry, The University of Southern California, Los Angeles, CA 90033, U.S.A. Hydrogen peroxide is cytotoxic to mammalian cells. The LDso for H20 2 in V79 Chinese hamster fibroblasts, Chinese hamster ovary cells and HA1 Chinese hamster fibroblasts is 2.0 p.Moles/107 cells, 7.0/zMoles/107 cells, and 4.4/zMoles/107 cells respectively. Although H20 2 is toxic we have found that if cells are pretreated with a small amount of H20 2 they can better withstand a later more severe H20 2 challenge. We have demonstrated an adaptive response to H20 2 in the Chinese hamster cell lines: V79, CHO, and HAl. Optimal adaptation was seen with HA1 cells which, when pretreated with 4.4 p.Moles H2Oz/107 cells exhibited a 25-fold increase in colony formation after being challenged with 11 p.Moles H202/107 cells; as compared to cells which were challenged but not pretreated. Adaptation was seen with pretreatment concentrations ranging from 2.5 /xMoles/107 cells to 5.5 /zMoles/107 cells. The time interval between pretreatment and challenge yielding optimal adaptation was 19 hours, with no adaptation appearing before 10 hours. Adaptation peaked at 19 hours and then declined, but was still evident after 22 hours. Cell density was critical for H202 treatment and cytotoxicity was linear with p.Moles of H202/cell rather than with the concentration of H202. Treatment with H202 caused an increase in cell size which was evident 5 hours after pretreatment with 4.4/zMoles of H202/107 cells. In addition, H202 was more cytotoxic when treatment was in complete media rather than in saline. At low levels, H202 actually stimulated colony formation. Ongoing work is aimed at determining the cause of these adaptations. Northern analysis of adapted cells using stress/shock probes will be presented. We are investigating the induction of HSP 70, GRP78, GRP94 and ERCC2. We will also present 2 dimensional electrophoresis analysis of proteins in adapted cells, as well as preliminary results of a Rot analysis and a partial subtractive hybridization.
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Oxidative Stress Genes and Proteins O X Y G E N R A D I C A L F O R M A T I O N BY E P I P H Y S E A L CHONDROCYTES CAUSES C H A N G E S IN C A R T I L A G E COLLAGEN SYNTHESIS Irving M. Shapiro, Toshikazu Tokuoka, Elizabeth Forbes, Kristine DeBolt and Manrizio Pacifici Departments of Biochemistry and Anatomy-Histology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, U.S.A. The epiphyseal growth plate is the site for the growth and elongation of bone. Our previous studies of this tissue indicate that reactive oxygen species (ROS) may be generated by cells in the epiphyseal hypertrophic region. To ascertain how ROS generation affects the synthesis of the extraeellular matrix proteins we set up a cell culture system to mimic events that occur in the development of the growth plate. Chondrocytes were isolated from the growth plates of chick embryo tibiae by a collagenase digestion procedure. The cells were cultured in a medium supplemented with ascorbate in the presence and absence of Fe(II). Measurements of thiobarbituric acid reactive substance (TBARS) production by these cultured chondroeytes showed that both ascorbate and Fe(II) activated ROS-dependent lipid peroxidation. Stimulation of ROS formation had a profound effect on protein synthesis. When the cells were labeled with [35]S-methionine for a short time period and the newly synthesized proteins were separated by PAGE in a 6% gel, changes were seen in a number of identified proteins; the most significant effect was an increase in the mobility of pro alpha Type II and Type X collagens. For both of these collagens, the lowered molecular weight was probably due to protein under-hydroxylation. It was concluded that ROS generation by cells of the epiphyseal growth cartilage can modify the chemical nature of the extracellular matrix by regulating the synthesis of proteins that are required for normal tissue development and mineralization.
1.23
YEAST AS A MODEL FOR OXIDANT INJURY. MECHANISM OF OXYGEN REGULATION OF ELECTRON TRANSPORT ACTIVITY Richard M. Wright, Webb-Waring Lung Institute at University of Colorado HSC,Denver, Colorado 80262 We are using the yeast Saccharomyces cerevisiae to model mechanisms of oxidant injury. The mitochondrial electron transport chain is potentially a major source of intracellular oxygen radicals, and its activity is strictly coupled to oxygen level. As oxygen tension falls activity of specific electron transport components falls. As a first step in our analyses, we have examined how oxygen regulates these changes inelectron transport activity. We find that hypoxic cultures are a) depressed in electron transport activity, b) show loss of the protein components at cytochrome aa3 (and elsewhere), e) have greatly decreased levels of steady state mRNAs for cytochrome aa3 genes, d) show decreased transcription of cytochrome aa3-1ac Z fusion plasmids, and e) the site conferring regulation by oxygen is the same as that conferring heme regulation of eytochrome aa3 genes. Our results suggest that the oxygen sensing mechanism for cytochrome aa3 is transduced through a heme dependent pathway as it is for cytochrome c. Interestingly, under conditions of hyperoxia we do not observe the corresponding effect at the level of transcription of cytoehrome aa3 genes or the cytochrome c gene. Perhaps in yeast hyperoxie injury is exerted entirely at the level of enzyme kinetics or activity and not at the level of gene expression.
IN SITU ANALYSIS OF MANGANESE SUPEROXIDE DISMUTASE (MnSOD) IN ALVEOLAR TYPE II CELLS OF HYPEROXIC RATS. Renaud Vincent, Ye-Shih Ho, Ling-Yi Chang, Jan W. Slot, and James D. Crapo. Duke University Medical Center, Durham, NC, 27710, and University of Utrecht, Utrecht, The Netherlands.
1.22
Rats exposed to 85% 0 2 for 5-7 days develop a tolerance to hyperoxia, which is manifested by prolonged survival in 100% O~, a treatment normally lethal within 60 hours. This tolerance m attributed to a selective increase of antioxidant defenses in the lungs, compensating for an higher intracellular flux of activated oxygen species. Total lung MnSOD is particularly increased following prolonged exposure to 85% 0 2. We have studied in situ by immunocytochemistry the time-cours~ and the magnitudeoft-~ee changes of MnSOD in alveolar type II cells, a probable locus of the antioxidant response. Rats were exposed to 85% Oafor 7 days and 14 days. The lungs were fixed, embedded in LP.-White, and sections for EM were labeled with anti-rat MnSOD antiserum followed by protein A-gold. Induction of MnSOD after 7 days was confirmed by the observation of llbroblasts and type II cells with high levels of the protein in their mitochondria. However, the response was not uniform. In average, the labeling density (gold/urn2) over mitochondria of type II cells increased by 22% above control value after 7 days, and 66% (p<0.05) after 14 days. The data support the concept of an increased concentration of MnSOD in the mitochondria, but the estimated contribution of type II cells to the total lung MnSOD response is mainly through hypertrophic and hyperplastic changes, and by an increased mitochondrial volume at 14 days (total type II cells MnSOD at 7 days, + 155% above controls; 14 days, + 413%). The steady-state level of mRNA for MnSOD in total lungs was found to increase rapidly during exposure to 85% O 2 (3 days) but returned to baseline by 14 days. These results contrasted with the time-course of enzyme increase, which appeared as a delayed event. We suggest that an early response of MnSOD in the lungs may be partly driven by inflammatory events, while the enzymatic changes measured at 14 days and the accumulation of mitochondria may constitute a direct oxygen effect, reflecting oxygen adaptation.
ADAPTIVE RESPONSE TO HYDROGEN PEROXIDE IN MAMMALIAN CELLS Anne G. Wiese, Robert E. Paciflci, and Kelvin J. A. Davies Institute for Toxicology & Department of Biochemistry, The University of Southern California, Los Angeles, CA 90033, U.S.A. Hydrogen peroxide is cytotoxic to mammalian cells. The LDso for H20 2 in V79 Chinese hamster fibroblasts, Chinese hamster ovary cells and HA1 Chinese hamster fibroblasts is 2.0 p.Moles/107 cells, 7.0/zMoles/107 cells, and 4.4/zMoles/107 cells respectively. Although H20 2 is toxic we have found that if cells are pretreated with a small amount of H20 2 they can better withstand a later more severe H20 2 challenge. We have demonstrated an adaptive response to H20 2 in the Chinese hamster cell lines: V79, CHO, and HAl. Optimal adaptation was seen with HA1 cells which, when pretreated with 4.4 p.Moles H2Oz/107 cells exhibited a 25-fold increase in colony formation after being challenged with 11 p.Moles H202/107 cells; as compared to cells which were challenged but not pretreated. Adaptation was seen with pretreatment concentrations ranging from 2.5 /xMoles/107 cells to 5.5 /zMoles/107 cells. The time interval between pretreatment and challenge yielding optimal adaptation was 19 hours, with no adaptation appearing before 10 hours. Adaptation peaked at 19 hours and then declined, but was still evident after 22 hours. Cell density was critical for H202 treatment and cytotoxicity was linear with p.Moles of H202/cell rather than with the concentration of H202. Treatment with H202 caused an increase in cell size which was evident 5 hours after pretreatment with 4.4/zMoles of H202/107 cells. In addition, H202 was more cytotoxic when treatment was in complete media rather than in saline. At low levels, H202 actually stimulated colony formation. Ongoing work is aimed at determining the cause of these adaptations. Northern analysis of adapted cells using stress/shock probes will be presented. We are investigating the induction of HSP 70, GRP78, GRP94 and ERCC2. We will also present 2 dimensional electrophoresis analysis of proteins in adapted cells, as well as preliminary results of a Rot analysis and a partial subtractive hybridization.
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