Genotoxicity of Electron Transport Inhibitors: Models of Neurodegenerative Diseases.





B. Van Houten, B.Mandavilli, J.Santos, L. Hunakova and Y. Chen.

Laboratory of Molecular Genetics, National Institute of Environmental and Health Sciences, National Institutes of Health, Research Triangle Park, NC- 27709



Using a quantitative PCR (QPCR) gene-specific assay we have shown that while mitochondria show good DNA repair capacity, protracted hydrogen peroxide treatment leads to extensive and persistent mitochondrial DNA (mtDNA) damage in a number of different primary and transformed cell lines. In order to investigate this phenomenon in more detail, we have studied hydrogen peroxide-induced DNA damage in human fibroblasts immortalized by expressing telomerase. We have found that hydrogen peroxide causes extensive mtDNA damage, with little or no nuclear damage. MtDNA damage was only repaired by 50% in 24 hrs. Cell sorting experiments using JC-1 showed that mtDNA damage persisted in cells with low membrane potential. These observations have lead to our group to test several hypotheses: 1) since mitochondrial DNA encodes 13 polypeptides, all of which are involved in electron transport or ATP production, loss of these proteins could lead to the generation of ROS and more mt DNA damage in a vicious feed-forward cascade; 2) several neurotoxicants that inhibit the electron transport (ET) could lead to the generation of ROS and subsequent mitochondrial DNA damage; and 3) mt DNA damage will be accumlate with age and in human diseases associated with reactive oxygen species, ROS. It has been suggested that Bcl-2 has anti-oxidant properties, while other studies have demonstrated that Bcl-2 acts to block the release of cytochrome C from mitochondria and activation of apoptosis. Using two rat PC12 cell lines, one of which over expresses Bcl-2, it was found that hydrogen peroxide or peroxynitrite induced more mtDNA damage than nuclear DNA damage in both cell lines. While hydrogen peroxide produced equal amounts of damage in the mtDNA in both cell lines, it was found that the cells over-expressing Bcl-2 repaired the mtDNA damage faster than the control parent line. This remarkable finding suggests that Bcl-2 protects mitochondria, not from the primary oxidative insult, but the secondary damage induced by mitochondrially-generated ROS. We have also examined the neurotoxicant, 3-nitropropionic acid, a complex II inhibitor that leads to the production of ROS, followed by mtDNA damage. Bcl-2 protects these cells from 3-NPA toxicity by protecting against the formation of ROS, and mtDNA damage. Gene expression profiling is being used to help understand these differences. A second neurotoxicant, MPTP, (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), which inhibits complex I inducing Parkinsonianism in humans and animals, lead to primarily mtDNA damage in dopaminergic neurons in mice. This finding supports the hypothesis that toxicants, which inhibit the electron transport, can lead to mitochondrially generated ROS and subsequent mtDNA damage, setting off a vicious cycle of damage and subsequent cell death.




Key words: DNA repair, mitochondrial DNA; oxidative stress; neurological degeneration







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