MOLECULAR MECHANISMS OF OXIDANT TOXICITY

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Aging is the highest risk factor for many fatal diseases. The Free Radical Theory of Aging argues for a role of oxidant toxicity in aging. Although oxidative damage accumulates during the process of aging, it is not clear how oxidants might cause aging at the mechanistic level. As the gene array technology evolves, it has been shown that tissues from old mice elevate stress responses and decrease metabolism by altering the expression of a large number of genes. Oxidants are known to induce stress responses and alterations of gene expression. In normal human diploid fibroblasts (HDFs) mild doses of oxidants cause the cells to develop a senescent phenotype prematurely. The phenotype switch suggests that multiple intrinsic changes may have been produced at the molecular level following oxidative stress. Elevation of p21 WAF17/Cip1/Sdi1 was found to precede the onset of the senescent phenotype, which involves an elevated expression of 8 senescence-associated genes. Preliminary studies using the microarray technology point to the direction that oxidants induce aging-associated genes as well as senescence- associated genes. These observations lead us to hypothesize that oxidants can induce the expression of aging-associated genes, some of which are controlled by p21. Mouse embryonic fibroblasts (MEFs) will allow us to determine the molecular program of oxidative stress that might be relevant to aging in vivo. Using the senescent phenotype as a marker of multiple molecular changes in MEFs, we will determine the gene expression pattern resulting from oxidative stress using a global and systematic approach involving gene array and Northern blot techniques. Dermal connective tissues from young and old mice will be compared to generate an aging gene profile. Comparison between the pattern of oxidative stress gene expression and the aging gene profile will allow us to critically test the relationship between oxidative stress and aging. This approach will also lead to the identification of important aging-associated changes that can be studied for their mechanisms of regulation at the cellular level. Since p21 has been reported to control the expression of a large number of genes, we will test the relationship between p21 and the expression of a limited number of functionally important genes shared by oxidative stress response and aging of dermal connective tissue using p21 knockout MEFs or HDFs. Finally, the mechanism of sustained p21 elevation following a pulse treatment of H2O2 will be determined by detailed analysis of transcriptional activation or mRNA stabilization. Because of the ubiquitousness of oxidants in our daily life, aging and disease states, it is important to understand the fundamental mechanisms of oxidant toxicity at the molecular level. We have a unique finding of premature senescence with oxidative stress and will combine in vitro and in vivo approaches to uncover the trigger of unwanted effects of aging.