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Superoxide-mediated cytotoxicity in superoxide dismutase-deficient fetal fibroblasts
T T Huang1, M Yasunami, E J Carlson
1Department of Pediatrics, University of California at San Francisco, 94143, USA. tthuang@itsa.ucsf.edu
Abstract:
To investigate the roles of CuZn superoxide dismutase (CuZnSOD) and Mn superoxide dismutase (MnSOD) in oxygen radical-mediated cytotoxicity and to distinguish the actions of these two enzymes, fetal fibroblasts were derived from mouse fetuses that are either deficient in CuZnSOD (Sod1-/+ and -/-) or MnSOD (Sod2-1+ and -/-) for in vitro studies. Whereas the phenotype of the Sod1 mutant animals did not differ from that of their normal littermates, the growth of Sod1-/- fetal fibroblasts was only 25% of that of the -/+ and +/+ cells. On the other hand, although almost all homozygous Sod2 mutant animals (-/-) died within 10 days after birth, cultivation of Sod2-/- fetal fibroblasts was possible and their growth was about 60% that of -/+ and +/+ cells. When cultured cells were subjected to treatment with paraquat to assess their ability to grow in the presence of high levels of superoxide radicals, Sod1-/- cells were 80 times more sensitive and Sod2-/- cells were 12 times more sensitive to paraquat than wild-type cells. In addition, whereas the loss of 50% CuZnSOD rendered Sod1-/+ cells almost twice more sensitive to paraquat than +/+ cells, loss of 50% MnSOD had no effect on paraquat sensitivity. Our results suggest that CuZnSOD-deficient cells are more sensitive to oxygen toxicity than are MnSOD-deficient cells, that paraquat causes free radical-induced damage in both the mitochondria and cytoplasm, and that SOD compartmentalized in the cytosol cannot compensate for the loss of SOD in the mitochondria and vice versa.
Insights
Mice lacking copper-zinc superoxide dismutase (CuZnSOD) showed impaired fibroblast growth and extreme sensitivity to oxygen radicals. Manganese superoxide dismutase (MnSOD) deficiency also impacted growth and sensitivity, but to a lesser extent.
Area of Science:
- Biochemistry
- Cell Biology
- Genetics
Background:
- Superoxide dismutases (SODs) are critical enzymes that neutralize harmful superoxide radicals.
- Two main isoforms, copper-zinc superoxide dismutase (CuZnSOD) and manganese superoxide dismutase (MnSOD), are found in different cellular compartments.
Purpose of the Study:
- To investigate the distinct roles of CuZnSOD and MnSOD in cellular defense against oxygen radical-mediated cytotoxicity.
- To differentiate the enzymatic actions and cellular localization effects of CuZnSOD and MnSOD.
Main Methods:
- Generation of mouse fetal fibroblasts deficient in either CuZnSOD (Sod1-/-) or MnSOD (Sod2-/-).
- In vitro cultivation and growth assessment of mutant and wild-type fibroblasts.
- Paraquat treatment to induce oxidative stress and evaluate cell sensitivity.
Main Results:
- Sod1-/- fibroblasts exhibited significantly reduced growth (25%) and extreme sensitivity (80x) to paraquat compared to wild-type.
- Sod2-/- fibroblasts showed reduced growth (60%) and increased sensitivity (12x) to paraquat.
- Partial loss of CuZnSOD (Sod1-/+) increased paraquat sensitivity, while partial MnSOD loss had no effect.
Conclusions:
- CuZnSOD deficiency confers greater sensitivity to oxygen toxicity than MnSOD deficiency.
- Paraquat induces damage in both mitochondrial and cytoplasmic compartments.
- SODs are compartmentalized, and cytosolic CuZnSOD cannot compensate for mitochondrial MnSOD loss, and vice versa.