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Elevated reactive oxygen species (ROS) and oxidative damage occur in the red blood cells (RBCs) of SOD1-deficient C57BL/6 mice. This leads to autoimmune responses against RBCs in aged mice that are similar to autoimmune hemolytic anemia (AIHA). We examined whether a SOD1 deficiency and/or the human SOD1 transgene (hSOD1) would affect phenotypes of AIHA-prone New Zealand Black (NZB) mice by establishing three congenic strains: those lacking SOD1, those expressing hSOD1 under a GATA-1 promoter, and those lacking mouse SOD1 but expressing hSOD1. Levels of intracellular ROS and oxidative stress markers increased, and the severity of the AIHA phenotype was aggravated by a SOD1 deficiency. In contrast, the transgenic expression of hSOD1 in an erythroid cell-specific manner averted most of the AIHA phenotype evident in the SOD1-deficient mice and also ameliorated the AIHA phenotype in the mice possessing intrinsic SOD1. These data suggest that oxidative stress in RBCs may be an underlying mechanism for autoimmune responses in NZB mice. These results were consistent with the hypothetical role of reactive oxygen species in triggering the autoimmune reaction in RBCs and may provide a novel approach to mitigating the progression of AIHA by reducing oxidative stress., (© 2013 Elsevier Inc. All rights reserved.)

Superoxide dismutase (SOD) plays a role in antioxidation, and SOD1-knockout (KO) mice show moderate phenotypes. Primary cultured mouse embryonic fibroblasts (MEFs) lead to growth failure and eventual death under normoxic culture (20% oxygen). We attempted to elucidate the molecular mechanisms responsible for the oxygen toxicity in SOD1-KO MEFs. Increases in reactive oxygen species, lipid peroxidation products, and senescence-associated β-galactosidase activity were observed in SOD1-KO MEFs. Hypoxic culture (2% oxygen) averted immediate cell death but could not recover the proliferative ability of the SOD1-KO cells. The cell cycles of SOD1-deficient MEFs were arrested at the G2 and M phases, leading to the accumulation of tetraploid cells under hypoxic culture. The suppressed expression of cyclin A2 and B1 and the concomitant induction of p21(Waf1) were evident in SOD1-KO cells. The phosphorylation of p53 and histone H2Ax and the induction of the two proapoptotic genes Bax and Noxa were evident in SOD1-deficient MEFs and more enhanced under normoxic culture than under hypoxic culture. We concluded that low levels of oxygen consumption moderately activates the p53 pathway, and leads to cellular senescence, but that high levels of oxygen consumption hyperactivates the p53 pathway, which results in cell death in SOD1-deficient MEFs., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Superoxide dismutase 1 (SOD1) is an important antioxidative enzyme that protects skin from oxidative stress. SOD1 (-/-) mice with a genetic background of b129Sv mice showed facial skin damage after 15 weeks of age. Eyelid swelling occurred as the initial symptom and caused impairment by triggering self-scratching. The period required for wound healing in the back was markedly delayed in 20-week SOD1 (-/-) mice. Oxidative stress markers, 4-hydroxynonenal and thiobarbituric acid-reactive substances, were unexpectedly lower in SOD1 (-/-) mice at day 1 after wounding. The decay rate of electron paramagnetic resonance signal intensity of intravenously injected nitroxide radical indicated that the half-life of the signal intensity was significantly prolonged in the wounded skin of SOD1 (+/+) mice. However, while the half-life of the signal intensity in control skin was a little longer in SOD1 (-/-) mice, it did not change in wounded skin. Taken together, these data suggest that the skin of SOD1 (-/-) mice is in redox imbalance and prone to damage by wounding.

We have recently shown that deficiency of the superoxide dismutase 1 gene (SOD1) causes anemia and autoimmune responses against red blood cells (RBCs) and that transgenic expression of human SOD1 in erythroid cells rescues them. Because these phenotypes observed in SOD1-deficient mice are similar to autoimmune hemolytic anemia (AIHA), a causal connection between reactive oxygen species (ROS) and AIHA was examined using an AIHA-prone New Zealand Black (NZB) mouse. ROS levels in RBCs were high in young NZB mice, compared to control New Zealand White (NZW) mice, and increased during aging. Methemoglobin and lipid peroxidation products were elevated during aging, consistent with the elevated oxidative stress in RBCs of NZB mice. Severity of anemia and levels of intracellular ROS were positively correlated. Levels of antibodies against 4-hydroxynonenal and acrolein were also elevated in NZB mice. Transgenic expression of human SOD1 protein in RBCs of NZB mice suppressed ROS in RBCs and decreased the death rate. When RBCs from C57BL/6 mice were injected weekly into the same strain of mice, production of anti-RBC antibody was observed only in mice that had been injected with oxidized RBCs. Thus, oxidation-mediated autoantibody production may be a more general mechanism for AIHA and related autoimmune diseases., (2010 Elsevier Inc. All rights reserved.)

Oxidative stress has been implicated as a cause of various diseases such as anaemia. We found that the SOD1 [Cu,Zn-SOD (superoxide dismutase)] gene deficiency causes anaemia, the production of autoantibodies against RBCs (red blood cells) and renal damage. In the present study, to further understand the role of oxidative stress in the autoimmune response triggered by SOD1 deficiency, we generated mice that had the hSOD1 (human SOD1) transgene under regulation of the GATA-1 promoter, and bred the transgene onto the SOD1(-/-) background (SOD1(-/-);hSOD1(tg/+)). The lifespan of RBCs, levels of intracellular reactive oxygen species, and RBC content in SOD1(-/-);hSOD1(tg/+) mice, were approximately equivalent to those of SOD1(+/+) mice. The production of antibodies against lipid peroxidation products, 4-hydroxy-2-nonenal and acrolein, as well as autoantibodies against RBCs and carbonic anhydrase II were elevated in the SOD1(-/-) mice, but were suppressed in the SOD1(-/-);hSOD1(tg/+) mice. Renal function, as judged by blood urea nitrogen, was improved in the transgenic mice. These results rule out the involvement of a defective immune system in the autoimmune response of SOD1-deficient mice, because SOD1(-/-);hSOD1(tg/+) mice carry the hSOD1 protein only in RBCs. Metabolomic analysis indicated a shift in glucose metabolism to the pentose phosphate pathway and a decrease in the energy charge potential of RBCs in SOD1-deficient mice. We conclude that the increase in reactive oxygen species due to SOD1 deficiency accelerates RBC destruction by affecting carbon metabolism and increasing oxidative modification of lipids and proteins. The resulting oxidation products are antigenic and, consequently, trigger autoantibody production, leading to autoimmune responses.

Prx (peroxiredoxin) is a multifunctional redox protein with thioredoxin-dependent peroxidase activity. Prx4 is present as a secretory protein in most tissues, whereas in sexually mature testes it is anchored in the ER (endoplasmic reticulum) membrane of spermatogenic cells via an uncleaved N-terminal hydrophobic peptide. We generated a Prx4 knockout mouse to investigate the function of Prx4 in vivo. Prx4(-/y) mice lacking Prx4 expression in all cells were obtained by mating Prx4(flox/+) female mice with Cre-transgenic male mice that ubiquitously expressed Cre recombinase. The resulting Prx4(-/y) male mice were fertile, and most organs were nearly normal in size, except for testicular atrophy. The number of deoxynucleotidyl transferase-mediated dUTP nick end labelling-positive spermatogenic cells was higher in Prx4(-/y) mice than in Prx4(+/y) mice and increased remarkably in response to warming the lower abdomen at 43 degrees C for 15 min. Cells reactive to antibodies against 4-hydroxynonenal and 8-hydroxyguanine were high in the Prx4(-/y) mice and concomitant with elevated oxidation of lipid and protein thiols. The cauda epididymis of Prx4(-/y) mice contained round spermatocytes, which were not found in Prx4(+/y) mice, and displayed oligozoospermia. However, mature spermatozoa from the epididymis of Prx4(-/y) mice exhibited normal fertilization In vitro. Taken together, these results indicate that spermatogenic cells lacking Prx4 are more susceptible to cell death via oxidative damage than their wild-type counterparts. Our results suggest that the presence of Prx4, most likely the membrane-bound form, is important for spermatogenesis, but not an absolute requisite.

Because glutathione scavenges reactive oxygen species (ROS) and also donates electrons to antioxidative systems, it may compensate for the oxidative stress caused by SOD1 deficiency. The cystine/glutamate transporter, which consists of two proteins, xCT and 4F2hc, has been designated system x (c) (-) . This transporter system plays a role in the maintenance of glutathione levels in mammalian cells. In the present study, we created SOD1 (-/-); xCT (-/-) double-knockout mice by intercrossing xCT-knockout and SOD1-knockout animals. We determined if the double-knockout mice express the phenotypic characteristics unique to SOD1 (-/-) mice-increased oxidative stress and the production of autoantibodies against erythrocytes. We also compared the phenotype of the double-knockout mice with those of the single-knockout and wild-type mice. Although two major antioxidative systems were found to be defective in the SOD1 (-/-); xCT (-/-) mice, relative to the SOD1 (-/-) mice, no functional deficits were observed. Based on these results, it appears that defects in system x (c) (-) do not exacerbate the phenotypic consequences of SOD1 deficiency in postnatal mice under ordinary breeding conditions.

The estrogen synthetase (aromatase, cytochrome P-450AROM) gene has been isolated from human genomic libraries and characterized. The restriction map of 43 positive clones obtained indicated that this enzyme is present as a single copy gene. The aromatase gene is unexpectedly large compared with other forms of the cytochrome P-450 superfamily, spanning at least 70 kilobases. The gene consists of 10 exons and its 5'-untranslated region is divided into 2 exons by an intron of more than 35 kilobases long. This organization of the first exon in the aromatase gene is unique in the cytochrome P-450 superfamily. All the exon-intron junctional sequences conform to the canonical GT/AG rule. The sequences of a TATA box and a CAAT box are present 27 and 83 base pairs upstream from the transcriptional initiation site. Within 3 kilobases upstream from the initiation site, there are no typical consensus sequences of responsive elements for glucocorticoid and c-AMP, which regulate aromatase expression.

Blue Cross and Blue Shield of Michigan assessed the intensity of inpatient laboratory use for similar patients in a sample of 30 short-term hospitals in three geographic areas. Hospital records were abstracted for 1,834 patients treated in 1980 for uncomplicated (Stage 1) admissions involving eight common diagnoses. It was found that patients treated in osteopathic teaching hospitals received significantly more laboratory tests than similar patients in allopathic teaching or nonteaching hospitals. Statistically significant differences were also found among the three geographic areas in the sample.