An assay for superoxide dismutase activity in mammalian tissue homogenates

Self Cite

Retinoids are signaling molecules that are involved in proliferation, differentiation and apoptosis during development. Retinoids exert their effects, in part, by binding to nuclear receptors, thereby altering gene expression. Clinical use of retinoids in the treatment of neuroblastoma is of interest due to their success in management of acute promyelocytic leukemia. Using the SK-N-SH human neuroblastoma cell line we investigated the effect of the differentiation agent, all-trans-retinoic acid (ATRA) on manganese superoxide dismutase (MnSOD) expression, an enzyme previously shown to enhance differentiation in vitro. Manganese superoxide dismutase mRNA, protein and activity levels increased in a time dependent manner upon treatment with ATRA. Nuclear levels of the NFκB proteins, p50 and p65, increased within 24 h of ATRA administration. This increase paralleled the degradation of the cytoplasmic inhibitor, IκB-β. Furthermore an increase in DNA binding activity to a NFκB element occurred within a 342 base pair enhancer (I2E) of the SOD2 gene with 10 μM ATRA treatment. Reporter analysis showed that ATRA-mediated I2E dependent luciferase expression was attenuated upon mutation of the NFκB element, suggesting a contribution of this transcription factor in retinoid-mediated upregulation of MnSOD. This study identifies SOD2 to be a retinoid responsive gene and demonstrates activation of the NFκB pathway in response to ATRA treatment of SK-N-SH cells. These results suggest signaling events involving NFκB and SOD2 may contribute to the effects of retinoids used in cancer therapy.

Section: Atra Increases Mnsod Activity In a Time Dependent Mannermentioning

confidence: 80%

Section: Superoxide Dismutase Activity Assaymentioning

confidence: 99%

All-trans-retinoic acid induces manganese superoxide dismutase in human neuroblastoma through NF-κB

Kiningham

Cardozo

Cook

et al. 2008

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“…Manganese superoxide dismutase (MnSOD) activity was measured by a modified nitroblue tetrazolium (NBT) assay, which is based on the competition scavenging of superoxide radicals, generated by xanthine/xanthine oxidase, between SOD and NBT [35]. Briefly, 20 μL of different amounts of total protein from heart tissue homogenate were added to the 160 μL of reaction buffer (50 mM potassium phosphate, pH 7.8, 1 mM diethylenetriaminepenta acetic (DETAPAC), 10 U/ml CAT, 56 μM NBT, 0.1 mM xanthine) in the presence of 5 mM NaCN, a copper-zinc-SOD inhibitor.…”

Section: Mnsod Activitymentioning

confidence: 99%

Phenylbutyrate, a histone deacetylase inhibitor, protects against Adriamycin-induced cardiac injury

Daosukho

Chen

Noel

et al. 2007

Cardiac injury is a major complication for oxidative stress-generating anticancer agents exemplified by Adriamycin (ADR). Recently, several histone deacetylase inhibitors (HDACIs) including phenylbutyrate (PBA) have shown promise in the treatment of cancer with little known toxicity to normal tissues. PBA has been shown to protect against oxidative stress in normal tissues. Here, we examined whether PBA might protect heart against ADR toxicity in a mouse model. The mice were i.p. injected with ADR (20 mg/kg). PBA (400 mg/kg/day) was i.p. injected one day before and daily after the ADR injection for two days. We found that PBA significantly decreased the ADR-associated elevation of serum lactase dehydrogenase (LDH) and creatine kinase (CK) activities, and diminished ADR-induced ultrastructual damages of cardiac tissue by more than 70%. Importantly, PBA completely rescued ADR-caused reduction of cardiac functions exemplified by ejection fraction and fraction shortening, and increased cardiac MnSOD protein and activity. Our results reveal a previously unrecognized role of HDACIs in protecting against ADR-induced cardiac injury, and suggest that PBA may exert its cardioprotective effect, in part, by the increase of MnSOD. Thus, combining HDACIs with ADR could add a new mechanism to fight cancer while simultaneously decrease ADR-induced cardiotoxicity.

“…Total superoxide dismutase (SOD) activity was measured by monitoring the reduction of cytochrome c at 550 nm in a reaction mixture containing 50 mM potassium phosphate (pH 7.5), 0.1 mM xanthine, 0.5 munit/ml xanthine oxidase, 0.1 mM EDTA and 10 μM cytochrome c. One unit of SOD represents the enzyme activity that causes a 50% inhibition in the reduction of cytochrome c. Aliquots of samples were taken to determine MnSOD activity by the addition of 5 mM KCN to inhibit CuZnSOD activity [18]. CuZnSOD activity was evaluated by subtracting MnSOD activity from the total SOD activity.…”

Section: Enzyme Activity and Glutathione Analysesmentioning

confidence: 99%

High superoxide dismutase and low glutathione peroxidase activities in red blood cells predict susceptibility of lung cancer patients to radiation pneumonitis

Park

Ramnath

Yang

et al. 2007

Radiation pneumonitis is an unpredictable complication of radiotherapy for lung cancer and a condition which can cause significant morbidity. The ability to identify patients at a high risk of developing pneumonitis is critical, since it will enable the individualization of the treatment plan. Because the cytotoxic effect of radiation is propagated through ROS and ROS-driven oxidative stress, the role of anti-oxidant defense systems in radiation pneumonitis was investigated. Using the pneumonitis-sensitive C3H/HeN mice as a model, we demonstrated that the anti-oxidant response of the lung correlated well with that of RBC. We then proceeded to test whether differences of RBC anti-oxidant response would predict the pneumonitis development in patients. SOD, GPX, CAT activities and glutathione in RBC were measured at baseline and then weekly for 6 weeks of treatment in fifteen eligible patients receiving concurrent chemo-radiotherapy for unresectable stage III NSCLC. Striking differences were found in the anti-oxidant activities of RBC with respect to the pneumonitis development. Those who developed pneumonitis showed higher SOD and lower GPX activities at baseline compared to those who did not (3.7 vs. 6.8 unit/mg for median SOD, 16.5 vs. 10.7 nmol/min/mg for median GPX). The functional imbalance of SOD and GPX was displayed consistently throughout the treatment period. The sensitivity and specificity of pneumonitis prediction were further increased when the GPX/SOD ratio was analyzed (pre-treatment P = 0.0046). Our results provide a strong rationale to monitor SOD and GPX activities of RBC to identify patients who are at risk of developing pneumonitis, and to implement a strategy of increasing the GPX/SOD ratio in order to lower the risk.