Gsh

Ascorbic Acid

Vitamin E

Fig. 4. Levels of GSH, ascorbic acid, and vitamin E in the heart and skeletal muscle of 12-week-old male MnSOD transgenic (T) and control (C) mice. The levels of GSH and ascorbic acid are expressed as micromoles per gram tissue, and that of vitamin E (»-tocopherol) as micrograms per gram tissue. The data are shown as means ± standard deviation (n = 6).

for speedy completion of these assays, which is essential to minimize the oxidation of these compounds during sample processing. Measurement of «-tocopherol by HPLC with fluorescence detection is simple and specific, and is much more sensitive than UV detection. The levels of GSH, ascorbic acid, and vitamin E (a-tocopherol) in the heart and skeletal muscle of MnSOD transgenic and control mice are shown in Fig. 4.

Implications

Free radical-induced oxidative damage may occur when antioxidant potential is decreased and/or when oxidative stress is increased.31 As a critical antioxidant enzyme in aerobic organisms, increased expression of MnSOD gene is expected to provide increased protection against oxidative stress. Indeed, intratracheal injection of adenovirus containing the human MnSOD gene protects athymic nude mice from irradiation-induced organizing alveolitis,32 and transgenic mice

32 M. W. Epperly, J. A. Bray, S. Krager, L. M. Berry, W. Gooding, J. F. Engelhardt, R. Zwacka, E. L. Travis, and J. S. Greenberger, Int. J. Radiat. Oncol. Biol. Phys. 43, 169 (1999).

expressing MnSOD in pulmonary epithelial cells are more resistant to oxygen toxicity.8 Also, transgenic mice expressing human MnSOD in the heart are protected against doxorubicin (Adriamycin)-induced cardiac toxicity9 and myocardial ischemia-reperfusion injury.33 In addition, expression of human MnSOD gene prevents neural apoptosis and reduces ischemic brain injury (suppression of peroxynitrite production, lipid peroxidation, and mitochondrial dysfunction)34 and attenuates l-methyl-4-phenyl-l,2,5,6-tetrahydropyridine (MPTP) toxicity35 in MnSOD transgenic mice. On the other hand, MnSOD knockout mice on a CD1 (outbred) genetic background die within the first 10 days of life 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 activities in the heart and, to a lesser extent, in other organs.6,36 Also, heterozygous MnSOD knockout mice with a 50% decrease in MnSOD activity, and no change in GSH peroxidase or Cu,ZnSOD activities, survive hyperoxia as well as their normal littermates, and do not develop any ultrastructural abnormality in the myocardium after 100% oxygen exposure for 90 hr.36,37 However, these heterozygous MnSOD knockout mice have increased hepatic mitochondrial oxidative damage and altered mitochondrial function. These findings indicate that MnSOD is required to maintain the integrity of mitochondrial enzymes susceptible to direct inactivation by superoxide, and that transgenic mice expressing the human MnSOD gene are protected under oxidative stress-mediated pathogenic conditions.

Induction of antioxidant enzymes, including MnSOD, in mammalian cells and tissues is generally accompanied by an increased tolerance to environmental agents that cause oxidative stress.38,39 However, as hydrogen peroxide is the dismutation product of SOD, it has been hypothesized that increased expression of MnSOD may enhance mitochondrial production of hydrogen peroxide and increase oxidative damage. This concern is promoted by the findings that overexpression of Cu,ZnSOD is associated with the rapid aging feature of the brains of patients with Down's syndrome,40,41 and that the pathogenic mechanisms in the motor

33 Z. Chen, B. Siu, Y. S. Ho, R. Vincent, C. C. Chua, R. C. Hamdy, and B. H. Chua, J. Mol. Cell Cardiol. 30, 2281 (1998).

34 J. N. Keller, M. S. Kindy, F. W. Holtsberg, D. K. St. Clair, H. C. Yen, A. Germeyer, S. M. Steiner, A. J. Bruce-Keller, J. B. Hutchins, and M. P. Mattson, J. Neurosci. 18,687 (1998).

35 P. Klivenyi, D. K. St. Clair, M. Wermer, H. C. Yen, T. Oberley, L. Yang, and M. Flint-Beal, Neurobiol. Dis. 5,253 (1998).

36 M. D. Williams, H. Van Remmen, C. C. Conrad, T. T. Huang, C. J. Epstein, and A. Richardson, J. Biol. Chem. 273, 28510 (1998).

37 M.F.Tsan, J. E. White, B. Caska, C. J. Epstein, and C. Y. Lee, Am. J. Cell Mol. Biol. 19,114(1998).

39 L. B. Clerch and D. Massaro, J. Clin. Invest. 91,499 (1993).

40 O. Elroy-Stein and Y. Groner, Cell 52, 259 (1988).

411. Ceballen-Picot, A. Nicole, P. Briand, G. Grimber, A. Delacourte, A. Defossez, F. Javoy-Agid, M. Lafon, J. L. Blouin, and P. M. Sinet, Brain Res. 552, 198 (1991).

neuron disease familial amyotrophic lateral sclerosis are associated with mutation of Cu,ZnSOD.4243 An enhancement of free radical formation due to a decrease in Km for hydrogen peroxide is linked to a gain-of-function of a familial amyotrophic lateral sclerosis-associated Cu,ZnSOD mutant. The reason for the contrasting effects of expression between the MnSOD gene and the Cu,ZnSOD gene is not clear. However, it appears that where oxygen radical is generated and the enzyme present may be critical in determining whether the expression of SOD is beneficial or detrimental.3

In the transgenic mice employed in our studies,9'34'35 heart and skeletal muscle exhibited the most significant amount of human MnSOD expression of all organs. Also, lung and brain had appreciable amounts of expression, whereas the expression in kidney and liver was limited.44 The activity of Cu,ZnSOD was significantly higher in kidney, but in other tissues analyzed, whereas catalase activity was significantly lower in brain and liver of transgenic mice than in those of their nontransgenic littermates. The activities of Se-GSH peroxidase and non-Se-GSH peroxidase were not significantly different among transgenic and nontransgenic mice in all tissues analyzed. Also, increased expression of human MnSOD genes did not significantly alter the levels of low molecular weight antioxidants GSH, ascorbic acid, and «-tocopherol in all tissues measured.

In agreement with the findings that transgenic mice expressing MnSOD are resistant to free radical-induced tissue injury,9 34-35 transgenic mice had significantly lower levels of lipid peroxidation product, mainly malondialdehyde, in the heart and muscle than did their nontransgenic littermates.44 The levels of conjugated dienes and protein carbonyls were not significantly different in tissues that overexpress the human MnSOD gene (heart, muscle, brain, and lung) or in nonover-expressed tissues (liver and kidney) between transgenic and nontransgenic mice. These findings suggest that the activities of GSH peroxidase and catalase present in various tissues of MnSOD transgenic mice are capable of handling the hydrogen peroxide generated and that small changes in the activities of Cu,ZnSOD and catalase in various tissues of MnSOD transgenic mice do not adversely affect their ability to handle hydrogen peroxide generated or influence the oxidative stress status. It also suggests that expression of the human MnSOD gene provides protection against peroxidative damage to membrane lipids, which may contribute to the increased resistance of MnSOD transgenic mice to the toxic effects of environmental agents. Thus, MnSOD transgenic mice, along with other transgenic mice expressing higher or lower levels of expression of antioxidant enzyme(s), should

42 M. E. Gurney, F. B. Cutting, P. Zhai, P. K. Andrus, and E. D. Hall, Pathol. Biol. 44, 51 (1996).

43 M. B. Yim, J. H. Kang, H. S. Yim, H. S. Kwak, P. B. Chock, and E. R. Stadtman, Proc. Natl. Acad.

44 W. Ibrahim, U.-S. Lee, H.-C. Yen, D. K. St. Clair, and C. K. Chow, Free Radic. Biol. Med. 28, 397

be a useful tool for unraveling the identity of reactive oxygen species that cause or promote the pathogenesis of various degenerative disorders and for defining the role of each antioxidant enzyme in cellular defense against free radical-mediated tissue injury.

Acknowledgments

Supported in part by NIH Grant CA80152.

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