Chloroplast Autophagy and Ubiquitination Combine to Manage Oxidative Damage and Starvation Responses
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Plant Sciences↗
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Oxidant damage during and after spaceflight
The objectives of this study were to assess oxidant damage during and after spaceflight and to compare the results against bed rest with 6 degrees head-down tilt. We measured the urinary excretion of the F(2) isoprostane, 8-iso-prostaglandin (PG) F(2alpha), and 8-oxo-7,8-dihydro-2 deoxyguanosine (8-OH DG) before, during, and after long-duration spaceflight (4-9 mo) on the Russian space station MIR, short-duration spaceflight on the shuttle, and 17 days of bed rest. Sample collections on MIR were obtained between 88 and 186 days in orbit. 8-iso-PGF(2alpha) and 8-OH DG are markers for oxidative damage to membrane lipids and DNA, respectively. Data are mean +/- SE. On MIR, isoprostane levels were decreased inflight (96. 9 +/- 11.6 vs. 76.7 +/- 14.9 ng. kg(-1). day(-1), P < 0.05, n = 6) due to decreased dietary intake secondary to impaired thermoregulation. Isoprostane excretion was increased postflight (245.7 +/- 55.8 ng. kg(-1). day(-1), P < 0.01). 8-OH DG excretion was unchanged with spaceflight and increased postflight (269 +/- 84 vs 442 +/- 180 ng. kg(-1). day(-1), P < 0.05). On the shuttle, 8-OH DG excretion was unchanged in- and postflight, but 8-iso-PGF(2alpha) excretion was decreased inflight (15.6 +/- 4.3 vs 8.0 +/- 2.7 ng. kg(-1). day(-1), P < 0.05). No changes were found with bed rest, but 8-iso-PGF(2alpha) was increased during the recovery phase (48.9 +/- 23.0 vs 65.4 +/- 28.3 ng. kg(-1). day(-1), P < 0.05). The changes in isoprostane production were attributed to decreased production of oxygen radicals from the electron transport chain due to the reduced energy intake inflight. The postflight increases in the excretion of the products of oxidative damage were attributed to a combination of an increase in metabolic activity and the loss of some host antioxidant defenses inflight. We conclude that 1) oxidative damage was decreased inflight, and 2) oxidative damage was increased postflight.
Mir Project↗
Transgenic Mouse Model for Reducing Oxidative Damage in Bone
Bone loss can occur due to many challenges such age, radiation, microgravity, and Reactive Oxygen Species (ROS) play a critical role in bone resorption by osteoclasts (Bartell et al. 2014). We hypothesize that suppression of excess ROS in skeletal cells, both osteoblasts and osteoclasts, regulates skeletal growth and remodeling. To test our hypothesis, we used transgenic mCAT mice which overexpress the human anti-oxidant catalase gene targeted to the mitochondria, the main site for endogenous ROS production. mCAT mice have a longer life-span than wildtype controls and have been used to study various age-related disorders. To stimulate remodeling, 16 week old mCAT mice or wildtype mice were exposed to treatment (hindlimb-unloading and total body-irradiation) or sham treatment conditions (control). Tissues were harvested 2 weeks later for skeletal analysis (microcomputed tomography), biochemical analysis (gene expression and oxidative damage measurements), and ex vivo bone marrow derived cell culture (osteoblastogenesis and osteoclastogenesis). mCAT mice expressed the transgene and displayed elevated catalase activity in skeletal tissue and marrow-derived osteoblasts and osteoclasts grown ex vivo. In addition, when challenged with treatment, bone tissues from wildtype mice showed elevated levels of malondialdehyde (MDA), indicating oxidative damage) whereas mCAT mice did not. Correlation analysis revealed that increased catalase activity significantly correlated with decreased MDA levels and that increased oxidative damage correlated with decreased percent bone volume (BVTV). In addition, ex-vivo cultured osteoblast colony growth correlated with catalase activity in the osteoblasts. Thus, we showed that these transgenic mice can be used as a model to study the relationship between markers of oxidative damage and skeletal properties. mCAT mice displayed reduced BVTV and trabecular number relative to wildtype mice, as well as increased structural model index in the cancellous tibia. Treatment caused bone loss in wildtype mice, as expected. Treatment also caused deficits in microarchitecture of mCAT mice, although less severe than wildtype mice in some parameters (percent bone volume, structural model index and cortical area). In conclusion, our results indicate that endogenous ROS signaling in both osteoblast and osteoclast lineage cells contributes to skeletal growth and remodeling, and quenching oxidative damage could play a role in bone loss prevention.
transgenic mice↗
Risk of Oxidative Damage to Bone from Increased Iron Stores During Space Flight
Iron stores are increased secondary to neocytolysis of red blood cells and a high dietary intake of iron during space flight. This raises concerns about the risk of excess iron causing oxidative damage in many tissues, including bone. Biomarkers of iron status, oxidative damage, and bone resorption during space flight were analyzed for 23 (16 M/7 F) International Space Station crewmembers as part of the Nutrition SMO project. Up to 5 in-flight blood samples and 24-h urine pools were collected over the course of the 4-6 month missions. Serum iron increased slightly during space flight and was decreased at landing (P < 0.0004). An increase in serum ferritin early in flight (217% in women and 68% in men, P < 0.0004), returning to preflight concentrations at landing, and a decrease in transferrin and transferrin receptors during flight indicated that a transient increase in iron stores occurred. No inflammatory response was observed during flight. The oxidative damage markers 8-hydroxy-2'-deoxyguanosine and prostaglandin F(sub 2(alpha)) were positively correlated (both P < 0.001) with serum ferritin. A greater area under the curve for ferritin during flight was correlated with greater changes in bone mineral density of several bone regions after flight (1). In a separate study (2), a ground-based investigation was conducted that examined the combined effects of radiation exposure and iron overload on sensitivity to radiation injury in several physiological systems in 12-wk male Sprague-Dawley rats. The rats were acclimated to an adequate iron diet (45 mg iron (ferric citrate)/kg diet) for 3 wk and then assigned to one of four groups: adequate iron (Fe) diet/no radiation, adequate Fe diet/ radiation, moderately high Fe diet (650 mg Fe (ferric citrate)/kg diet)/no radiation, and moderately high Fe diet/radiation. Animals remained on the assigned diet for 4 wk. Starting on day 14 of experimental diet treatment, animals were exposed to a fractionated dose (0.375 Gy) of Cs-137 every other day (3 Gy total dose). On day 29 (24 h after last radiation exposure), animals were euthanized. Oxidative stress markers in the liver, bone, eyes, and serum were assessed. There was evidence that the iron diet contributed to DNA damage as well as radiation exposure in the liver, eyes, and bone. Together, the results suggest that increased iron stores do constitute a risk factor for oxidative damage and bone resorption, during space flight and on Earth. Funded by the Human Health and Countermeasures Element of the NASA Human Research Program.
Zwart, S. R.↗
Predicting oxidation damage of ultra high-temperature carbide ceramics in extreme environments using machine learning
Determining the oxidation resistance of UHTC carbides in extreme environments is challenging theoretically and experimentally due to the high dimensional complexity of influencing variables and intricate testing setups. Herein we demonstrate the use of machine learning (ML) models trained with experimental literature data to predict the oxide thickness of UHTC carbides exposed to air based on composition, mean grain size, relative densification, holding time, and temperature. A multi-dimensional database with 76 occurrences is created containing experimental results of Hf, Zr, and Ta carbides plus additives. In this study, the preprocessed database is then used to train ML models to predict their oxidation behavior. The trained model predicts the oxidation damage in the form of an average oxide thickness in UHTC carbides with a Mean Absolute Error (MAE) of ±65.45 μm for samples in the testing set that developed thicknesses up to 1000 μm. The model successfully predicted oxidation damage for a recession rate lower than 60 μm/min. It is noticed that the ensemble method MAE is increased to ±134.34 μm while forecasting the oxidation of samples with a recession rate higher than the threshold. The unprecedented approach is a novel way to predict the damage through the oxidation of carbide compounds before processing for a smarter design with room for improvement.
36 MATERIALS SCIENCE↗
Identification of the prooxidant site of human ceruloplasmin: a model for oxidative damage by copper bound to protein surfaces
Free transition metal ions oxidize lipids and lipoproteins in vitro; however, recent evidence suggests that free metal ion-independent mechanisms are more likely in vivo. We have shown previously that human ceruloplasmin (Cp), a serum protein containing seven Cu atoms, induces low density lipoprotein oxidation in vitro and that the activity depends on the presence of a single, chelatable Cu atom. We here use biochemical and molecular approaches to determine the site responsible for Cp prooxidant activity. Experiments with the His-specific reagent diethylpyrocarbonate (DEPC) showed that one or more His residues was specifically required. Quantitative [14C]DEPC binding studies indicated the importance of a single His residue because only one was exposed upon removal of the prooxidant Cu. Plasmin digestion of [14C]DEPC-treated Cp (and N-terminal sequence analysis of the fragments) showed that the critical His was in a 17-kDa region containing four His residues in the second major sequence homology domain of Cp. A full length human Cp cDNA was modified by site-directed mutagenesis to give His-to-Ala substitutions at each of the four positions and was transfected into COS-7 cells, and low density lipoprotein oxidation was measured. The prooxidant site was localized to a region containing His426 because CpH426A almost completely lacked prooxidant activity whereas the other mutants expressed normal activity. These observations support the hypothesis that Cu bound at specific sites on protein surfaces can cause oxidative damage to macromolecules in their environment. Cp may serve as a model protein for understanding mechanisms of oxidant damage by copper-containing (or -binding) proteins such as Cu, Zn superoxide dismutase, and amyloid precursor protein.
NASA Discipline Regulatory Physiology↗
NDE for Characterizing Oxidation Damage in Reinforced Carbon-Carbon
In this study, coated reinforced carbon-carbon (RCC) samples of similar structure and composition as that from the NASA space shuttle orbiter s thermal protection system were fabricated with slots in their coating simulating craze cracks. These specimens were used to study oxidation damage detection and characterization using NDE methods. These specimens were heat treated in air at 1143 and 1200 C to create cavities in the carbon substrate underneath the coating as oxygen reacted with the carbon and resulted in its consumption. The cavities varied in diameter from approximately 1 to 3 mm. Single-sided NDE methods were used since they might be practical for on-wing inspection, while x-ray micro-computed tomography (CT) was used to measure cavity sizes in order to validate oxidation models under development for carbon-carbon materials. An RCC sample having a naturally-cracked coating and subsequent oxidation damage was also studied with x-ray micro-CT. This effort is a follow-on study to one that characterized NDE methods for assessing oxidation damage in an RCC sample with drilled holes in the coating. The results of that study are briefly reviewed in this article as well. Additionally, a short discussion on the future role of simulation to aid in these studies is provided.
Roth, Don J.↗
Nondestructive Evaluation (NDE) for Characterizing Oxidation Damage in Cracked Reinforced Carbon-Carbon
In this study, coated reinforced carbon-carbon (RCC) samples of similar structure and composition as that from the NASA space shuttle orbiter's thermal protection system were fabricated with slots in their coating simulating craze cracks. These specimens were used to study oxidation damage detection and characterization using nondestructive evaluation (NDE) methods. These specimens were heat treated in air at 1143 C and 1200 C to create cavities in the carbon substrate underneath the coating as oxygen reacted with the carbon and resulted in its consumption. The cavities varied in diameter from approximately 1 to 3mm. Single-sided NDE methods were used because they might be practical for on-wing inspection, while X-ray micro-computed tomography (CT) was used to measure cavity sizes in order to validate oxidation models under development for carbon-carbon materials. An RCC sample having a naturally cracked coating and subsequent oxidation damage was also studied with X-ray micro-CT. This effort is a follow-on study to one that characterized NDE methods for assessing oxidation damage in an RCC sample with drilled holes in the coating.
Roth, Don J.↗
Nondestructive Evaluation (NDE) for Characterizing Oxidation Damage in Cracked Reinforced Carbon-Carbon (RCC)
In this study, coated reinforced carbon-carbon (RCC) samples of similar structure and composition as that from the NASA space shuttle orbiter's thermal protection system were fabricated with slots in their coating simulating craze cracks. These specimens were used to study oxidation damage detection and characterization using nondestructive evaluation (NDE) methods. These specimens were heat treated in air at 1143 and 1200 C to create cavities in the carbon substrate underneath the coating as oxygen reacted with the carbon and resulted in its consumption. The cavities varied in diameter from approximately 1 to 3 mm. Single-sided NDE methods were used since they might be practical for on-wing inspection, while x-ray micro-computed tomography (CT) was used to measure cavity sizes in order to validate oxidation models under development for carbon-carbon materials. An RCC sample having a naturally-cracked coating and subsequent oxidation damage was also studied with x-ray micro-CT. This effort is a follow-on study to one that characterized NDE methods for assessing oxidation damage in an RCC sample with drilled holes in the coating.
Roth, Don J.↗
Predicting oxidation damage in ultra high-temperature borides: A machine learning approach
Ultra-high temperature (UHT) borides are ceramics materials with melting points above 3000 °C for structural applications in extreme environments. However, at temperatures exceeding 1600 °C and under oxidizing conditions, the material suffers from detrimental degradation. Optimized design and performance of diboride materials under such extreme conditions requires filling the missing composition-microstructure-oxidation gap. This study proposes a computational data-driven framework to connect the processing and microstructure of Ultra-high temperature borides with the oxidation damage. Random Forest Regressor (RFR) model is adopted to forecast the oxide scale thickness developed after oxidation testing based on processing variables and microstructural features. The model trained on a dataset consisting of 107 samples of experimental data extracted from the literature aims to predict oxidation damage. With proper data manipulation and fine model tuning, the predictor could forecast the oxide scale thickness of UHT diborides with a Mean Absolute Error of 37.45 μm and an R-square of 0.83. This model could be used as a high-throughput scheme to design and test new UHT diborides materials computationally. Furthermore, a model with larger composition capabilities could also be developed in the future as more experimental data become available.
36 MATERIALS SCIENCE↗
Ultrasonic assessment of interfacial oxidation damage in ceramic matrix composites
A new approach to characterizing oxidation damage in ceramic matrix composites using ultrasonic techniques is proposed. In this approach, the elastic constants of the composite are determined nondestructively by measuring the angular dependence of both longitudinal and transverse wave velocities. A micromechanical model for composites with anisotropic constituents is used to find the anisotropic properties of an effective fiber, which is a combination of the fiber and the interface. Interfacial properties are extracted from the properties of this effective fiber by analyzing the difference between effective and actual fiber properties. Unidirectional /0/28 SiC/Si3N4 composites with 30 percent fiber volume fraction and 30 percent matrix porosity are used. The samples are exposed in a flowing oxygen environment at elevated temperatures, up to 1400 C, for 100 hours and then measured by ultrasonic methods at room temperature. The Young's modulus in the fiber direction of the sample oxidized at 600 C decreased significantly but it was unchanged for samples oxidized at temperatures above 1200 C. The transverse moduli obtained from ultrasonic measurements decrease continuously up to 1200 C. The shear stiffnesses show behavior similar to the transverse moduli. The effective elastic moduli of the interfacial carbon coating are determined from the experimental data, and their change due to thermal oxidation is discussed.
Chu, Y. C.↗
Processing of DNA single-strand breaks with oxidatively damaged ends by LIG1
Abstract DNA ligase 1 (LIG1) seals broken strand breaks by joining two adjacent ends during DNA replication and repair transactions. We previously reported atomic-level insight into the strategies that LIG1 uses to discriminate mismatches or ribonucleotides. However, how LIG1 processes strand breaks with oxidatively damaged ends in the absence and presence of a “wrong” sugar remains unknown. Here, we determined the crystal structures of LIG1/nick DNA complexes with 3′-8-oxodG and 3′-8-oxorG templating A or C during the pre- and post-catalytic steps of the ligation reaction. Our structures demonstrated differences in the distances at the +1 and +2 nucleotides relative to the 3′-end of the nick and a shift in the template base position to accommodate the oxidative lesion depending on the dual coding potential of 8-oxoG, which forms Hoogsteen or Watson–Crick base pairing in -syn or -anti conformation. Furthermore, these structural adjustments lead to mutagenic ligation or non-mutagenic end joining of the nick substrates. Overall, our findings provide mechanistic insight into how LIG1 processes nicks harboring oxidative damage and ribonucleotides to ensure fidelity at the final ligation step of DNA repair and replication to maintain genome integrity.
Balu, Kanal E [Department of Biochemistry and Mole↗
Protective Effect of Phloretin against Hydrogen Peroxide-Induced Oxidative Damage by Enhancing Autophagic Flux in DF-1 Cells
Phloretin (PHL) is a dihydrochalcone flavonoid isolated from the peel and root bark of apples, strawberries, and other plants with antioxidative characteristic. In this study, we aimed to investigate the protective effect and the potential mechanism of PHL on hydrogen peroxide (H2O2)-induced oxidative damage in DF-1 cells. The results showed that PHL exhibited no cytotoxic effect on DF-1 cells at concentration below 20 μM. PHL markedly increased H2O2-reduced cell viability, decreased H2O2-induced apoptosis, as evidenced by reduced apoptosis rate, the upregulation of gene and protein level of Bcl-2, and the downregulation of gene and protein level of Bax and Cleaved caspase3. In addition, PHL reduced H2O2-induced reactive oxygen species (ROS) production and restored antioxidant enzymes activities as well as mitochondrial membrane potential in a dose-dependent manner. Moreover, PHL prior to H2O2 further increased LC3-II level, promoted p62 turnover and improved lysosomal function. Importantly, autophagy inhibitor chloroquine (CQ) reversed the protective effect of PHL, and increased H2O2-induced apoptosis. Furthermore, PHL inhibited the phosphorylation levels of ERK, p38, and JNK. Collectively, these results indicate that PHL could attenuate H2O2-induced oxidative injury and apoptosis by maintaining lysosomal function and promoting autophagic flux, and MAPKs pathway may be involved in this process. Our study provides evidence that PHL could as a new strategy to against oxidative damage in poultry industry.
Song, Dan↗
Ceruloplasmin copper induces oxidant damage by a redox process utilizing cell-derived superoxide as reductant
Oxidative damage by transition metals bound to proteins may be an important pathogenic mechanism. Ceruloplasmin (Cp) is a Cu-containing plasma protein thought to be involved in oxidative modification of lipoproteins. We have previously shown that Cp increased cell-mediated low-density lipoprotein (LDL) oxidation by a process requiring cell-derived superoxide, but the underlying chemical mechanism(s) is (are) unknown. We now show that superoxide reduction of Cp Cu is a critical reaction in cellular LDL oxidation. By bathocuproine disulfonate (BCS) binding and by superoxide utilization, we showed that exogenous superoxide reduces a single Cp Cu atom, the same Cu required for LDL oxidation. The Cu atom remained bound to Cp during the redox cycle. Three avenues of evidence showed that vascular cells reduce Cp Cu by a superoxide-dependent process. The 2-fold higher rate of Cp Cu reduction by smooth muscle cells (SMC) compared to endothelial cells (EC) was consistent with their relative rates of superoxide release. Furthermore, Cp Cu reduction by cells was blocked by Cu,Zn superoxide dismutase (SOD1). Finally, the level of superoxide produced by EC and SMC was sufficient to cause the amount of Cu reduction observed. An important role of Cp Cu reduction in LDL oxidation was suggested by results showing that SOD1 inhibited Cp Cu reduction and LDL oxidation by SMC with equal potency, while tumor necrosis factor-alpha stimulated both processes. In summary, these results show that superoxide is a critical cellular reductant of divalent transition metals involved in oxidation, and that protein-bound Cu is a substrate for this reaction. The role of these mechanisms in oxidative processes in vivo has yet to be defined.
NASA Discipline Regulatory Physiology↗
Role of Oxidative Damage in Radiation-Induced Bone Loss
During prolonged spaceflight, astronauts are exposed to both microgravity and space radiation, and are at risk for increased skeletal fragility due to bone loss. Evidence from rodent experiments demonstrates that both microgravity and ionizing radiation can cause bone loss due to increased bone-resorbing osteoclasts and decreased bone-forming osteoblasts, although the underlying molecular mechanisms for these changes are not fully understood. We hypothesized that excess reactive oxidative species (ROS), produced by conditions that simulate spaceflight, alter the tight balance between osteoclast and osteoblast activities, leading to accelerated skeletal remodeling and culminating in bone loss. To test this, we used the MCAT mouse model; these transgenic mice over-express the human catalase gene targeted to mitochondria, the major organelle contributing free radicals. Catalase is an anti-oxidant that converts reactive species, hydrogen peroxide into water and oxygen. This animal model was selected as it displays extended lifespan, reduced cardiovascular disease and reduced central nervous system radio-sensitivity, consistent with elevated anti-oxidant activity conferred by the transgene. We reasoned that mice overexpressing catalase in mitochondria of osteoblast and osteoclast lineage cells would be protected from the bone loss caused by simulated spaceflight. Over-expression of human catalase localized to mitochondria caused various skeletal phenotypic changes compared to WT mice; this includes greater bone length, decreased cortical bone area and moment of inertia, and indications of altered microarchitecture. These findings indicate mitochondrial ROS are important for normal bone-remodeling and skeletal integrity. Catalase over-expression did not fully protect skeletal tissue from structural decrements caused by simulated spaceflight; however there was significant protection in terms of cellular oxidative damage (MDA levels) to the skeletal tissue. Furthermore, we used an array of countermeasures (Antioxidant diets and injections) to prevent the radiation-induced bone loss, although these did not prevent bone loss, analysis is ongoing to determine if these countermeasure protected radiation-induced damage to other tissues.
oxidative damage↗
Detection of Strand Cleavage And Oxidation Damage Using Model DNA Molecules Captured in a Nanoscale Pore
We use a biological nano-scale pore to distinguish among individual DNA hairpins that differ by a single site of oxidation or a nick in the sugar-phosphate backbone. In earlier work we showed that the protein ion channel alpha-hemolysin can be used as a detector to distinguish single-stranded from double-stranded DNA, single base pair and single nucleotide differences. This resolution is in part a result of sensitivity to structural changes that influence the molecular dynamics of nucleotides within DNA. The strand cleavage products we examined here included a 5-base-pair (5-bp) hairpin with a 5-prime five-nucleotide overhang, and a complementary five-nucleotide oligomer. These produced predictable shoulder-spike and rapid near-full blockade signatures, respectively. When combined, strand annealing was monitored in real time. The residual current level dropped to a lower discrete level in the shoulder-spike blockade signatures, and the duration lengthened. However, these blockade signatures had a shorter duration than the unmodified l0bp hairpin. To test the pore sensitivity to nucleotide oxidation, we examined a 9-bp hairpin with a terminal 8-oxo-deoxyguanosine (8-oxo-dG), or a penultimate 8-oxo-dG. Each produced blockade signatures that differed from the otherwise identical control 9bp hairpins. This study showed that DNA structure is modified sufficiently by strand cleavage or oxidation damage at a single site to alter in a predictable manner the ionic current blockade signatures produced. This technique improves the ability to assess damage to DNA, and can provide a simple means to help characterize the risks of radiation exposure. It may also provide a method to test radiation protection.
Vercoutere, W.↗
Towards an Understanding of Oxidative Damage in an a-L-Arabinofuranosidase of Trichoderma reesei : a Molecular Dynamics Approach
Trichoderma reesei is a ‘workhorse’ fungus that produces glycosyl hydrolases (e.g., cellulases) at high titers for use in industrial bioprocessing. In this study, we focused on two side-chain hemicellulases, a-L-arabinofuranosidases and a-D-galactosidases, which are important for the treatment of lignocellulosic biomass, but susceptible to oxidative damage that can occur during industrial processing. The molecular details that render these enzymes inactive have not yet been identified. To approach this issue, we used proteomics to identify amino acid residues that were oxidized after a relevant oxidative treatment (Fenton reaction). These oxidative modifications were included in the 3D-protein structures, and using molecular dynamics simulations, we then studied the behaviors of non-modified and oxidized enzymes. These simulations showed significant alterations of the conformational stability of proteins when oxidized, as evidenced by changes in root mean square deviation (RMSD) and principal component analyses (PCA) trajectories. Likewise, enzyme-ligand interactions such as hydrogen bonds were greatly reduced in quantity and quality in the oxidized proteins. Finally, free energy landscape plots showed that there was a more rugged energy surface in the oxidized proteins, implying a less favorable reaction pathway. These results reveal the basis for loss of function in these carbohydrate active enzymes (CAZYs) in T. reesei.
60 APPLIED LIFE SCIENCES↗
Transgenic Mouse Model for Reducing Oxidative Damage in Bone
Exposure to musculoskeletal disuse and radiation result in bone loss; we hypothesized that these catabolic treatments cause excess reactive oxygen species (ROS), and thereby alter the tight balance between bone resorption by osteoclasts and bone formation by osteoblasts, culminating in bone loss. To test this, we used transgenic mice which over-express the human gene for catalase, targeted to mitochondria (MCAT). Catalase is an anti-oxidant that converts the ROS hydrogen peroxide into water and oxygen. MCAT mice were shown previously to display reduced mitochondrial oxidative stress and radiosensitivity of the CNS compared to wild type controls (WT). As expected, MCAT mice expressed the transgene in skeletal tissue, and in marrow-derived osteoblasts and osteoclast precursors cultured ex vivo, and also showed greater catalase activity compared to wildtype (WT) mice (3-6 fold). Colony expansion in marrow cells cultured under osteoblastogenic conditions was 2-fold greater in the MCAT mice compared to WT mice, while the extent of mineralization was unaffected. MCAT mice had slightly longer tibiae than WT mice (2%, P less than 0.01), although cortical bone area was slightly lower in MCAT mice than WT mice (10%, p=0.09). To challenge the skeletal system, mice were treated by exposure to combined disuse (2 wk Hindlimb Unloading) and total body irradiation Cs(137) (2 Gy, 0.8 Gy/min), then bone parameters were analyzed by 2-factor ANOVA to detect possible interaction effects. Treatment caused a 2-fold increase (p=0.015) in malondialdehyde levels of bone tissue (ELISA) in WT mice, but had no effect in MCAT mice. These findings indicate that the transgene conferred protection from oxidative damage caused by treatment. Unexpected differences between WT and MCAT mice emerged in skeletal responses to treatment.. In WT mice, treatment did not alter osteoblastogenesis, cortical bone area, moment of inertia, or bone perimeter, whereas in MCAT mice, treatment increased these parameters. Taken together, this typically catabolic treatment (disuse and irradiation) appeared to stimulate cortical expansion in MCAT mice but not WT mice. In conclusion, these results reveal the importance of mitochondrial ROS generation in skeletal remodeling and show that MCAT mice provide a useful animal model for bone studies.
trandgenic↗