Ting-Ting Huang

CuZnSOD deficiency leads to persistent and widespread oxidative damage and hepatocarcinogenesis later in life

Mice deficient in CuZn superoxide dismutase (CuZnSOD) showed no overt abnormalities during development and early adulthood, but had a reduced lifespan and increased incidence of neoplastic changes in the liver. Greater than 70% of Sod1−/− mice developed liver nodules that were either nodular hyperplasia or hepatocellular carcinoma (HCC). Cross-sectional studies with livers collected from Sod1−/− and age-matched +/+ controls revealed extensive oxidative damage in the cytoplasm and, to a lesser extent, in the nucleus and mitochondria from as early as 3 months of age. A marked reduction in cytosolic aconitase, increased levels of 8-oxo dG and F2-isoprostanes, and a moderate reduction in glutathione peroxidase activities and porin levels were observed in all age groups of Sod1−/− mice examined. There were also age-related reductions in Mn superoxide dismutase activities and carbonic anhydrase III. Parallel to the biochemical changes, there were progressive increases in the DNA repair enzyme APEX1, the cell cycle control proteins cyclin D1 and D3, and the hepatocyte growth factor receptor Met. Increased cell proliferation in the presence of persistent oxidative damage to macromolecules likely contributes to hepatocarcinogenesis later in life.

Increased Oxidative Damage Is Correlated to Altered Mitochondrial Function in Heterozygous Manganese Superoxide Dismutase Knockout Mice

This study characterizes mitochondria isolated from livers of Sod2 −/+ andSod2 +/+ mice. A 50% decrease in manganese superoxide dismutase (MnSOD) activity was observed in mitochondria isolated from Sod2 −/+ mice compared withSod2 +/+ mice, with no change in the activities of either glutathione peroxidase or copper/zinc superoxide dismutase. However, the level of total glutathione was 30% less in liver mitochondria of the Sod2 −/+ mice. The reduction in MnSOD activity in Sod2 −/+ mice was correlated to an increase in oxidative damage to mitochondria: decreased activities of the Fe-S proteins (aconitase and NADH oxidoreductase), increased carbonyl groups in proteins, and increased levels of 8-hydroxydeoxyguanosine in mitochondrial DNA. In contrast, there were no significant changes in oxidative damage in the cytosolic proteins or nuclear DNA. The increase in oxidative damage in mitochondria was correlated to altered mitochondrial function. A significant decrease in the respiratory control ratio was observed in mitochondria isolated from Sod2 −/+ mice compared with Sod2 +/+ mice for substrates metabolized by complexes I, II, and III. In addition, mitochondria isolated from Sod2 −/+ mice showed an increased rate of induction of the permeability transition. Therefore, this study provides direct evidence correlating reduced MnSOD activity in vivo to increased oxidative damage in mitochondria and alterations in mitochondrial function.

Alterations in mitochondrial function, hydrogen peroxide release and oxidative damage in mouse hind-limb skeletal muscle during aging.

Mitochondrial function, hydrogen peroxide generation and oxidative damage were measured in hind-limb skeletal muscle from young (6-8 month) and old (27-29 month) wildtype and heterozygous Mn-superoxide dismutase (MnSOD) knockout mice (Sod2(+/-)). The reduction in MnSOD activity in the Sod2(+/-) mice makes these mice a good model to examine the implications of life-long elevated endogenous mitochondrial oxidative stress on mitochondrial function. ATP production was reduced approximately 30% with age in skeletal muscle mitochondria isolated from wildtype mice, and reduced 40-45% in mitochondria from both young and old Sod2(+/-) mice compared to the young wildtype mice. Release of hydrogen peroxide from skeletal muscle mitochondria increased 40-50% with age in both wildtype and Sod2(+/-) but was not higher in mitochondria from Sod2(+/-) mice. Activities of electron transport Complexes I and V were decreased 25-30% in both young and old Sod2(+/-) mice compared to wildtype mice, and were 25-30% lower in mitochondria from old wildtype and old Sod2(+/-) mice. DNA oxidative damage (oxo8dG levels) increased more than 45% with age and over 130% in the young Sod2(+/-) mice compared to the wildtype mice. These data show that mitochondrial oxidative stress in mouse skeletal muscle is increased with age, leading to alterations in mitochondrial function. In addition, increased oxidative stress generated by reduced activity of MnSOD does not exacerbate these alterations during aging.

Genetic Dissection of Region Associated with Behavioral Abnormalities in Mouse Models for Down Syndrome

Two animal models of Down syndrome (human trisomy 21) with segmental trisomy for all (Ts65Dn) or part (Ts1Cje) of human chromosome 21-homologous region of mouse chromosome 16 have cognitive and behavioral abnormalities. To compare these trisomies directly and to assess the phenotypic contribution of the region of difference between them, Ts65Dn, Ts1Cje, and a new segmental trisomic (Ms1Ts65) for the region of difference (App to Sod1) have been generated as littermates and tested in parallel. Although the performance of Ts1Cje mice in the Morris water maze is similar to that of Ts65Dn mice, the reverse probe tests indicate that Ts65Dn is more severely affected. By contrast, the deficits of Ms1Ts65 mice are significantly less severe than those of Ts65Dn. Therefore, whereas triplication of Sod1 to Mx1 plays the major role in causing the abnormalities of Ts65Dn in the Morris water maze, imbalance of App to Sod1 also contributes to the poor performance. Ts65Dn mice are hyperactive and Ts1Cje mice are hypoactive; the activity of Ms1Ts65 mice is not significantly above normal. These findings indicate that genes in the Ms1Ts65 trisomic region must interact with others in the Ts1Cje region to produce hyperactivity in Ts65Dn mice.

Manganese superoxide dismutase signals matrix metalloproteinase expression via H2O2-dependent ERK1/2 activation.

Manganese-superoxide dismutase (Sod2) removes mitochondrially derived superoxide (O⨪2) at near-diffusion limiting rates and is the only antioxidant enzyme whose expression is regulated by numerous stimuli. Here it is shown thatSod2 also serves as a source of the intracellular signaling molecule H2O2.Sod2-dependent increases in the steady-state levels of H2O2 led to ERK1/2 activation and subsequent downstream transcriptional increases in matrix metalloproteinase-1 (MMP-1) expression, which were reversed by expression of the H2O2-detoxifying enzyme, catalase. In addition, a single nucleotide polymorphism has recently been identified (1G/2G) at base pair −1607 that creates an Ets site adjacent to an AP-1 site at base pair −1602 and has been shown to dramatically enhance transcription of the MMP-1 promoter. Luciferase promoter constructs containing either the 1G or 2G variation were 25- or 1000-fold more active when transiently transfected intoSod2-overexpressing cell lines, respectively. The levels of MMP-2, -3, and -7 were also increased in theSod2-overexpressing cell lines, suggesting thatSod2 may function as a “global” redox regulator of MMP expression. In addition, Sod2 − /+mouse embryonic fibroblasts failed to respond to the cytokine-mediated induction of the murine functional analog of MMP-1, MMP-13. This study provides evidence that the modulation of Sod2 activity by a wide array of pathogenic and inflammatory stimuli may be utilized by the cell as a primary signaling mechanism leading to matrix metalloproteinase expression.

Genetic modification of prenatal lethality and dilated cardiomyopathy in Mn superoxide dismutase mutant mice.

Mn superoxide dismutase (MnSOD), a mitochondrial antioxidant enzyme, has been shown to be essential for animal survival. MnSOD mutant mice (Sod2-/- mice) on the CD1 background develop severe dilated cardiomyopathy and usually die within 10 d after birth. To characterize better the phenotype and understand the mechanism of superoxide-mediated tissue damage in Sod2-/- mice, congenic Sod2-/- mice on inbred backgrounds were generated to ensure genetic homogeneity. When generated on a C57BL/6J background (B6), more than half of the fetuses develop severe dilated cardiomyopathy by embryonic day 15 and die in the uterus. Those that survive to term usually die within 24 h. In contrast, Sod2-/- mice on DBA/2J (D2) and B6D2F1 (B6D2F1) backgrounds develop normally throughout gestation and do not develop dilated cardiomyopathy. However, the D2 mice do develop a severe metabolic acidosis and survive for only up to 12 d after birth. B6D2F1) mice have a milder form of metabolic acidosis and can survive for up to 3 weeks. The marked difference in lifespans and the development of dilated cardiomyopathy in the B6 but not the D2 or B6D2F1 backgrounds indicate the possible existence of genetic modifiers that provide protection to the developing hearts in the absence of MnSOD.

The Use of Transgenic and Mutant Mice to Study Oxygen Free Radical Metabolism

ABSTRACT: To distinguish the role of Mn superoxide dismutase (MnSOD) from that of cytoplasmic CuZn superoxide dismutase (CuZnSOD), the mouse MnSOD gene (Sod2) was inactivated by homologous recombination. Sod2−/− mice on a CD1 (outbred) genetic background die within the first 10 days of life (mean, 5.4 days) with a complex phenotype that includes dilated cardiomyopathy, accumulation of lipid in liver and skeletal muscle, metabolic acidosis and ketosis, and a severe reduction in succinate dehydrogenase (complex II) and aconitase (a TCA cycle enzyme) activities in the heart and, to a lesser extent, in other organs. These findings indicate that MnSOD is required to maintain the integrity of mitochondrial enzymes susceptible to direct inactivation by superoxide. On the other hand, Lebovitz et al. reported an independently derived MnSod null mouse (Sod2tmlLeb) on a mixed C57BL/6 and 129Sv background with a different phenotype. Because a difference in genetic background is the most likely explanation for the phenotypic differences, the two mutant lines were crossed into different genetic backgrounds for further analyses. To study the phenotype of Sod2tmlLeb mice CD1 background, the Sod2tmlLeb mice were crossed to CD1 for two generations before the −/+ mice were intercrossed to generate −/− mice. The life span distribution of CD1〈Sod2−/−〉Leb was shifted to the left, indicating a shortened life span on the CD1 background. Furthermore, the CD1〈Sod2−/−〉Leb mice develop metabolic acidosis at an early stage as was observed with CD1〈Sod2−/−〉Cje. When Sod2tmlCje was placed on C57BL/6J (B6) background, the −/− mice were found to die either during midgestation or within the first 4 days after birth. However, when the B6〈Sod2−/+〉Cje were crossed with DBA/2J (D2) for the generation of B6D2F2〈Sod2−/−〉Cje mice, an entirely different phenotype, similar to that described by Lebovitz et al., was observed. The F2 Sod−/− mice were able to survive up to 18 days, and the animals that lived for more than 15 days displayed neurological abnormalities including ataxia and seizures. Their hearts were not as severely affected as were those of the CD1 mice, and neurological degeneration rather than heart defect appears to be the cause of death.

Manganese superoxide dismutase deficiency enhances cell turnover via tumor promoter-induced alterations in AP-1 and p53-mediated pathways in a skin cancer model

Previous studies in our laboratories demonstrated that overexpression of manganese superoxide dismutase (MnSOD) suppressed both the incidence and multiplicity of papillomas in a DMBA/TPA multi-stage skin carcinogenesis model. The activity of activator protein-1 (AP-1), which is associated with tumor promotion, was reduced in MnSOD transgenic mice overexpressing MnSOD in the skin, suggesting that MnSOD may reduce tumor incidence by suppressing AP-1 activation. In the present study, we report that reduction of MnSOD by heterozygous knockout of the MnSOD gene (Sod2 −/+, MnSOD KO) increased the levels of oxidative damage proteins and the activity of AP-1 following TPA treatment. RNA levels of ornithine decarboxylase (ODC) were also increased, suggesting an increase in cell proliferation in the KO mice. Histological examination confirmed that the number of proliferating cells in DMBA/TPA-treated mouse skin were higher in the KO mice. Interestingly, histological examination also demonstrated greater numbers of apoptotic cells in the KO mice after DMBA/TPA treatment. Evidence of apoptosis, including DNA fragmentation, cytochrome c release from mitochondria, and caspase 3 activation were also observed by biochemical assays of the skin tissues. Apoptosis was associated with an increase in nuclear levels of p53 as determined by Western analysis. Quantitative immunogold ultrastructural analysis confirmed that p53 immunoreactive protein levels were increased to a greater level in the nuclei of epidermal cells from MnSOD KO mice compared to epidermal nuclei from wild type mice similarly treated. Moreover, p53 levels further increased in the mitochondria of DMBA/TPA treated mice, and this increase was much greater in the MnSOD KO than in the wild type mice, suggesting a link between MnSOD deficiency and mitochondrial-mediated apoptosis. Pathological examination reveals no difference in the incidence and frequency of papillomas comparing the KO mice and their wild type littermates. Taken together, these results suggest that: (1) MnSOD deficiency enhanced TPA-induced oxidative stress and AP-1 and p53 levels, consistent with the increase in both proliferation and apoptosis events in the MnSOD KO mice, and (2) increased apoptosis may negate increased proliferation in the MnSOD deficient mice during an early stage of tumor development.

Strain-dependent high-level expression of a transgene for manganese superoxide dismutase is associated with growth retardation and decreased fertility.

Manganese superoxide dismutase (MnSOD) is essential in protecting mitochondria against the damaging effects of superoxide radicals (O(2)(*-)), and increased expression of MnSOD protects cells and transgenic animals from various forms of oxidative stress. In addition, increased levels of MnSOD have been shown to slow down cell growth and induce differentiation. To study the effects of high MnSOD levels in vivo, we generated a series of transgenic mice using a mouse genomic sequence under control of the endogenous promoter. Four transgenic lines produced by pronuclear DNA injection exhibited up to 2-fold elevated MnSOD levels in brain and heart. However, using an embryonic stem cell approach, a line having 10-fold elevated MnSOD levels in the brain and 6- to 7-fold elevated levels in the heart and kidney was generated. Surprisingly, the genetic background of this transgenic line influenced the expression level of the transgene, with DBA/2 (D2) and C57BL/6 (B6) mice exhibiting low- and high-level transgene expression, respectively. This difference was the result of an increased transcription rate of the transgene. High-level MnSOD expression in B6 animals was associated with small size, male infertility, and decreased female fertility. These features are absent on the D2 background and indicate that high levels of MnSOD activity may interfere with normal growth and fertility.

Oxidative stress and adult neurogenesis – effects of radiation and superoxide dismutase deficiency

Hippocampus plays an important role in learning and memory and in spatial navigation. Production of new neurons that are functionally integrated into the hippocampal neuronal network is important for the maintenance of functional plasticity. In adults, production of new neurons in the hippocampus takes place in the subgranular zone (SGZ) of dentate gyrus. Neural progenitor/stem cells go through processes of proliferation, differentiation, migration, and maturation. This process is exquisitely sensitive to oxidative stress, and perturbation in the redox balance in the neurogenic microenvironment can lead to reduced neurogenesis. Cranial irradiation is an effective treatment for primary and secondary brain tumors. However, even low doses of irradiation can lead to persistent elevation of oxidative stress and sustained suppression of hippocampal neurogenesis. Superoxide dismutases (SODs) are major antioxidant enzymes for the removal of superoxide radicals in different subcellular compartments. To identify the subcellular location where reactive oxygen species (ROS) are continuously generated after cranial irradiation, different SOD deficient mice have been used to determine the effects of irradiation on hippocampal neurogenesis. The study results suggest that, regardless of the subcellular location, SOD deficiency leads to a significant reduction in the production of new neurons in the SGZ of hippocampal dentate gyrus. In exchange, the generation of new glial cells was significantly increased. The SOD deficient condition, however, altered the tissue response to irradiation, and SOD deficient mice were able to maintain a similar level of neurogenesis after irradiation while wild type mice showed a significant reduction in the production of new neurons.