Significance of AtMTM1 and AtMTM2 for Mitochondrial MnSOD Activation in Arabidopsis

The manganese (Mn) tracking factor for mitochondrial Mn superoxide dismutase (MnSOD) has been annotated as yMTM1 in yeast, which belongs to the mitochondrial carrier family. We confirmed that Arabidopsis AtMTM1 and AtMTM2 are functional homologs of yMYM1 as they can revive yeast MnSOD activity in yMTM1-mutant cells. Transient expression of AtMnSOD-3xFLAG in the AtMTM1 and AtMTM2-double mutant protoplasts confirmed that AtMTM1 and AtMTM2 are required for AtMnSOD activation. Our study revealed that AtMnSOD interacts with AtMTM1 and AtMTM2 in the mitochondria. The expression levels of AtMTM1, AtMTM2, and AtMnSOD respond positively to methyl viologen (MV) and metal stress. AtMTM1 and AtMTM2 are involved in Mn and Fe homeostasis, root length, and flowering time. Transient expression of chloroplast-destined AtMnSOD revealed that an evolutionarily conserved activation mechanism, like the chloroplastic-localized MnSOD in some algae, still exists in Arabidopsis chloroplasts. This study strengthens the proposition that AtMTM1 and AtMTM2 participate in the AtMnSOD activation and ion homeostasis.

58 The Effect of Altering Dietary Manganese and Selenium Levels on the Growth Performance and Blood Manganese-superoxide Dismutase Activity in Nursery Pigs

The objective of this study was to determine the effect of altering dietary levels of manganese and selenium on growth performance and MnSOD specific activity. Weaned piglets were blocked by weight (n = 216; 5.21 ± 1.17 kg; 21 ± 3 d) and sex (2 barrows and 2 gilts/pen). Pens within a block were randomly assigned to dietary treatments in a factorial design with main effects of Se (0.1 and 0.3 ppm) and Mn (0, 12, and 24 ppm). Diets (n = 9 pens/treatment) were fed in three phases (P1 = d 1–7, P2 = d 7–21, P3 = d 21–35). Pigs and orts were weighed weekly and pigs were bled on d 0, 7, 21, and 35 for analysis of specific activity of red blood cell MnSOD. Data were analyzed as 2x3 factorial design via SAS PROC GLM. There were no Se effects or Mn x Se interactions observed. There was a linear increase (P < 0.05) in overall ADG (397, 424, and 438 g day-1pig-1 for 0, 12, 24 ppm Mn, respectively). There was a linear trend (P < 0.1) in overall ADFI (557, 560, 592 g day-1pig-1 for 0, 12, 24 ppm Mn, respectively). Feed efficiency (d 0–35) across increasing Mn levels were 0.71, 0.76, 0.74 (P > 0.1). MnSOD activity in RBC increased from d 0–7, peaked on d 7 and decreased to d 35. On d 7, diets with 12 and 0 ppm Mn had significantly increased (P < 0.05) MnSOD activity (0.91 and 0.87 IU mg-1 protein, respectively) compared to diets with 24 ppm Mn (0.67 IU mg-1 protein). The dietary treatment that best maintained MnSOD activity was 0.3 ppm Se and 12 ppm Mn supplemented, while ADG was maximized in the diet with 0.3 ppm Se and 24 ppm Mn supplementation.

Activity of Antioxidant Enzymes in the Tumor and Adjacent Noncancerous Tissues of Non-Small-Cell Lung Cancer

Lung tissue is directly exposed to high oxygen pressure, as well as increased endogenous and exogenous oxidative stress. Reactive oxygen species (ROS) generated in these conditions play an important role in the initiation and promotion of neoplastic growth. In response to oxidative stress, the antioxidant activity increases and minimizes ROS-induced injury in experimental systems. The aim of the present study was to evaluate the activity of antioxidant enzymes, such as superoxide dismutase (SOD; isoforms: Cu/ZnSOD and MnSOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione S-transferase (GST), along with the concentration of malondialdehyde (MDA) in tumor and adjacent noncancerous tissues of two histological types of NSCLC, i.e., adenocarcinoma and squamous cell carcinoma, collected from 53 individuals with surgically resectable NSCLC. MDA concentration was similar in tumors compared with adjacent noncancerous tissues. Tumor cells had low MnSOD activity, usually low Cu/ZnSOD activity, and almost always low catalase activity compared with those of the corresponding tumor-free lung tissues. Activities of GSH-related enzymes were significantly higher in tumor tissues, irrespective of the histological type of cancer. This pattern of antioxidant enzymes activity could possibly be the way by which tumor cells protect themselves against increased oxidative stress.

Activity of manganese superoxide dismutase (MnSOD) as a predictor of radiation therapy outcome in patients with stage IIIB squamous cell carcinoma cervical cancer

BACKGROUND Radiation is a standard therapy for cervical cancer. Unfortunately, not all patients undergoing radiation achieve a complete response. Previous studies have shown that manganese superoxide dismutase (MnSOD) acts against free radicals generated by radiation in cancer cells thus predicting worse outcome in radiation therapy. This study was aimed to assess and evaluate whether MnSOD activity can be used as a predictor of radiation therapy responses in patients with stage IIIB squamous cell carcinoma (SCC). METHODS A comparative cross-sectional study was conducted in the Gynecology Oncology Division, Department of Obstetrics and Gynecology, Faculty of Medicine, Universitas Indonesia, Cipto Mangunkusumo Hospital, Jakarta. The database from previous research was analyzed to identify positive and negative response samples. Measurement of MnSOD activity was done using spectrophotometry based on the McCord and Fridovich method using RanSOD® kit. The comparative data were obtained and then analyzed. RESULTS Among 76 samples, 49 (61.8%) patients had positive responses and 27 (38.2%) had negative responses. It is shown in this study that higher MnSOD activity is related to worse radiotherapy outcome in stage IIIB cancer patients. The relative risk value of having a worse outcome with high MnSOD activity is 1.849 (1.075–3.178, 95% CI). CONCLUSIONS Stage IIIB squamous cell carcinoma patients with high MnSOD activity are at higher risk of having a negative radiation therapy response compared with patients without high MnSOD activity.

PCB11 Metabolite, 3,3’-Dichlorobiphenyl-4-ol, Exposure Alters the Expression of Genes Governing Fatty Acid Metabolism in the Absence of Functional Sirtuin 3: Examining the Contribution of MnSOD

Although the production of polychlorinated biphenyls (PCBs) is prohibited, the inadvertent production of certain lower-chlorinated PCB congeners still threatens human health. We and others have identified 3,3’-dichlorobiphenyl (PCB11) and its metabolite, 3,3’-dichlorobiphenyl-4-ol (4OH-PCB11), in human blood, and there is a correlation between exposure to this metabolite and mitochondrial oxidative stress in mammalian cells. Here, we evaluated the downstream effects of 4OH-PCB11 on mitochondrial metabolism and function in the presence and absence of functional Sirtuin 3 (SIRT3), a mitochondrial fidelity protein that protects redox homeostasis. A 24 h exposure to 3 μM 4OH-PCB11 significantly decreased the cellular growth and mitochondrial membrane potential of SIRT3-knockout mouse embryonic fibroblasts (MEFs). Only wild-type cells demonstrated an increase in Manganese superoxide dismutase (MnSOD) activity in response to 4OH-PCB11–induced oxidative injury. This suggests the presence of a SIRT3-mediated post-translational modification to MnSOD, which was impaired in SIRT3-knockout MEFs, which counters the PCB insult. We found that 4OH-PCB11 increased mitochondrial respiration and endogenous fatty-acid oxidation-associated oxygen consumption in SIRT3-knockout MEFs; this appeared to occur because the cells exhausted their reserve respiratory capacity. To determine whether these changes in mitochondrial respiration were accompanied by similar changes in the regulation of fatty acid metabolism, we performed quantitative real-time polymerase chain reaction (qRT-PCR) after a 24 h treatment with 4OH-PCB11. In SIRT3-knockout MEFs, 4OH-PCB11 significantly increased the expression of ten genes controlling fatty acid biosynthesis, metabolism, and transport. When we overexpressed MnSOD in these cells, the expression of six of these genes returned to the baseline level, suggesting that the protective role of SIRT3 against 4OH-PCB11 is partially governed by MnSOD activity.

Use of molecular biomarkers to estimate manganese requirements for broiler chickens from 22 to 42 d of age

The present study was carried out to evaluate dietary Mn requirements of broilers from 22 to 42 d of age using molecular biomarkers. Chickens were fed a conventional basal maize–soyabean meal diet supplemented with Mn as Mn sulphate in graded concentrations of 20 mg Mn/kg from 0 to 140 mg Mn/kg of diet for 21 d (from 22 to 42 d of age). The Mn response curves were fitted for ten parameters including heart Mn-containing superoxide dismutase (MnSOD) mRNA and its protein expression levels and the DNA-binding activities of specificity protein 1 (Sp1) and activating protein-2 (AP-2). Heart MnSOD mRNA and protein expression levels showed significant quadratic responses (P<0·01), and heart MnSOD activity showed a broken-line response (P<0·01), whereas Mn content and DNA-binding activities of Sp1 and AP-2 in the heart displayed linear responses (P<0·01) to dietary Mn concentrations, respectively. The estimates of dietary Mn requirements were 101, 104 and 94 mg/kg for full expressions of MnSOD mRNA level, MnSOD protein level and MnSOD activity in the heart, respectively. Our findings indicate that heart MnSOD mRNA expression level is a more reliable indicator than heart MnSOD protein expression level and its activity for the evaluation of Mn requirement of broilers, and about 100 mg Mn/kg of diet is required for the full expression of heart MnSOD in broilers fed the conventional basal maize–soyabean meal diet from 22 to 42 d of age.

Dysfunctional MnSOD leads to redox dysregulation and activation of prosurvival AKT signaling in uterine leiomyomas

AKT signaling promotes cell growth and survival and is often dysregulated via multiple mechanisms in different types of cancer, including uterine leiomyomas (ULMs). ULMs are highly prevalent fibrotic tumors that arise from the smooth muscular layer of the uterus, the myometrium (MM). ULMs pose a major public health issue because they can cause severe morbidity and poor pregnancy outcomes. ‬We investigate the mechanisms driving ULM growth and survival via aberrant activation of AKT. We demonstrate that an acetylation-mediated impairment of the manganese superoxide dismutase (MnSOD) activity is prevalent in ULM cells compared to the normal-matched MM from the same patients. This impairment increases the levels of superoxide and oxidative stress, which activate AKT via oxidative inactivation of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Redox activation of AKT promotes ULM cell survival under conditions of moderate but persistent oxidative stress that are compatible with ULM’s prooxidative microenvironment. Moreover, because of impaired MnSOD activity, ULM cells are sensitive to high levels of reactive oxygen species (ROS) and superoxide-generating compounds, resulting in decreased ULM cell viability. On the contrary, MM cells with functional MnSOD are more resistant to high levels of oxidants. This study demonstrates a causative role of acetylation-mediated MnSOD dysfunction in activating prosurvival AKT signaling in ULMs. The specific AKT and redox states of ULM cells provide a potential novel therapeutic rationale to selectively target ULM cells because of their defective ROS-scavenging system.‬‬‬‬‬‬‬‬

Abstract 446: Estrogen and Estrogen Receptor Protect the Female Heart From Ischemia/reperfusion Injury by Increasing the Activity of Mitochondrial p38β and MnSOD

Background: We have previously demonstrated a novel in-vitro mechanism of how 17β-estradiol (E2) protects cultured neonatal cardiomyocytes from H/R by identifying a functionally active mitochondrial pool of p38β and E2-driven upregulation of manganese superoxide (MnSOD) activity via p38β. This protective mechanism leads to E2-mediated suppression of reactive oxygen species and attenuates apoptosis. However, little is known about these cytoprotective actions of E2 in the heart after myocardial I/R in vivo . The aim of this study is to determine whether the cardioprotective effects of E2 involve the interaction of mitochondrial p38β and MnSOD in the heart in vivo . Methods and Results: Left coronary ligation for 45 min and reperfusion for 24 hr was used to produce myocardial I/R injury in ovariectomized female mice with or without E2 pellet (0.1 mg/pellet). TTC staining showed that the infarct size was approximately 30% of the left ventricle at risk in untreated mice after I/R. The infarct size was reduced by more than half in the E2-treated group. Western blotting showed that the phosphorylation of p38β (p-p38β) in the heart homogenate was upregulated by E2, compared to the untreated mice, while the total p38β protein level was not altered. Mitochondrial p-p38β isolated from the hearts decreased after I/R, while E2 administration attenuated this effect. The total protein level of MnSOD decreased after I/R, regardless of E2 treatment. However, the MnSOD activity was normalized by E2 treatment, suggesting that E2 specifically increased the enzymatically active subset of MnSOD. Physical interaction between mitochondrial p38β and MnSOD was found by using co-immunoprecipitation from the hearts of the studied animals. Estrogen receptor (ER) α, β and double knockout female mice exhibited larger infarct size after I/R injury than that of WT mice while no significant difference was observed between the knockout mice. Loss of both ERs led to reduced activity of mitochondrial p38β and MnSOD at the baseline and after myocardial I/R. Conclusion: This work offers in-vivo evidence to our previously detailed mechanistic data that the protective effects of estrogen and ER against cardiac I/R injury involve the regulation of MnSOD activity via mitochondrial p38β.