an-chored to the inner membrane and is not freely mobile inside the matrix space 64, 109, 131, 331. It is precisely this association to the inner membrane that makes mtDNA even more vul-nerable to oxidative damage, because of its proximity to the superoxide producing ETC.
Studies in isolated mitochondria have shown that complex III is the primary site for super-oxide release from the ETC. But there is evidence that supersuper-oxide is also released into the inter-membrane space 332, were it can be disproportionated by SOD1 200 (N.B. SOD1 is able to translocated under increased oxidative stress into the intermembrane space). The validity of these data has been challenged, particularly in view of the fact that the ROS formation
was often determined under non-physiological conditions 333. But two recent studies have shown that the production of superoxide also occurs in isolated mitochondria, maintained under very physiological conditions 334, 335. But these results indicate that production of superoxide originates mainly from complex I. In contrast to complex III, the produced superoxide by complex I is released only into the matrix space 336. Therefore it is likely that oxidative damage to mtDNA is triggered by superoxide released from complex I. Complex I in turn might be most prone to be affected by mtDNA mutations, as 7 out of the 13 polypeptides encoded by mtDNA belong to this complex 337. Under physiological condi-tions the mitochondrial respiratory chain converts approximately 0.1% of oxygen into ROS. Therefore, SOD2, which is restricted to mitochondria, is essential for cell survival as evidenced by the lethality of Sod2-/- mice 338. These animals die within a few days after birth and exhibit a variety of phenotypes including extensive mitochondrial dysfunction and damage, neurodegeneration and cardiovascular abnormalities. Several pathophysiological conditions such as inflammation or chronic hypertension are associated with a counter-regulatory increase in SOD2 to cope with increased ROS formation 339. The common polymorphism in the mitochondrial leader sequence, which supposedly decreases the mito-chondrial import of SOD2, is associated with increased risk of various forms of cancer such as breast or prostate carcinoma 340.
For many of the known nucleoid-associated proteins no clear function has been assigned 64,
110, 128. For SOD2 it is obvious to suggest that a close proximity of SOD2 to mtDNA could prevent superoxide toxicity, in which protein-bound FeIII and a subsequent Fenton-reaction with H2O2 yields •OH-radicals or a ferryl ion. Hydrogen peroxide should therefore be dele-terious as well and requires an enzymatic system for detoxification. This was confirmed by co-immunoprecipitation of SOD2 with an antibody against GPx1 and by Western blot analysis of sucrose density gradient fractions. The emerging concept of a functional anti-oxidant system established in the nucleoid structure is supported by the notion that free mtDNA is more vulnerable to X-ray or H2O2 induced damage than mtDNA organized in nucleoids 341. One may argue that dense packing alone may already result in such protec-tion, but it was found that in E. coli, SOD2 is directly associated with DNA and could pro-tect against superoxide whereas bacterial Fe-SOD showed no association and provided no protection 212. The evolutionary explanation for the bacterial origin of mitochondria is an-other argument for similarities in the association of SOD2 in the bacterial and mitochon-drial genome.
Indirect proof of an increased antioxidant capability of mtDNA associated versus free matrix-located SOD2 may be taken from a comparison between endothelial and smooth muscle cells, with endothelial cells displaying no association of SOD2 with mtDNA.
Smooth muscle cells were reported to be more resistant towards oxidative stress-induced mtDNA lesions than endothelial cells 342. Importantly, endothelial cells were also more sen-sitive to peroxynitrite mediated mtDNA damage compared to smooth muscle cells 343. It may be speculated that the rapidly proliferating mitochondrial network in the endothelium could be the cause for the absence of an association of SOD2 to mtDNA.
It is obvious that SOD2 in conjunction with GPx1 protects mtDNA from oxidative dam-age triggered by superoxide and mediated by hydroxyl radicals. But in contrast to the popu-lar notion that ROS are mainly responsible for oxidative DNA damage, the results obtained in the present work indicate that peroxynitrite could be much more important than previ-ously assumed. Experiments with a plasmid model showed that equimolar fluxes of •NO and •O2- are very efficient at generating 8-oxodG, whereas induction of strand breaks did not occur (chapter 4.2.1.1). In physiological concentrations (inside mitochondria), per-oxynitrite was able to induce oxidative lesions in a dose dependent manner. With the high-est concentration of the peroxynitrite producing compound Sin-1, lesions were generated that were sufficient for a nearly complete unwinding of the plasmid DNA in the FADU-assay. Superoxide dismutase 2 was able to completely prevent the 8-oxodG generation, pre-sumably by reaction with superoxideand thereby inhibiting peroxynitrite formation. The important role for SOD2 in preventing peroxynitrite formation was thereby shown. Hy-drogen peroxide which was formed by the SOD2 reaction had no influence on 8-oxodG formation as well as on generation of strand breaks. Also experiments with the hypoxan-thine/xanthine oxidase system showed no significant 8-oxodG formation, although the amounts of superoxide produced by this system obviously were beyond physiological rele-vance (chapter 4.2.1.2). Once again no effect of H2O2 was observed, as was revealed by adding SOD2 in order to disproportionate superoxide into H2O2. Thereby it was unambi-guously shown that superoxide as well as H2O2 are irrelevant for the generation of 8-oxodG.
5.3 New hypothesis for a vicious circle involved in the mitochondrial theory