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MitoQ counteracts telomere shortening and elongates lifespan of fibroblasts under mild oxidative stress

Oxidative damage is thought to be a major causal factor for replicative senescence and human aging (Harman, 1994). Leakage of superoxide from the mitochondrial respiratory chain is an important source of oxidative stress (Raha & Robinson, 2000). Targeting antioxidants to mitochondria is an efficient way to attenuate oxidative damage in mitochondria due to the production of reactive oxygen species (ROS) in isolated mitochondria (Kelso et al ., 2001; Echtay et al ., 2002) and in mitochondria within cells (Kelso et al ., 2001; Hwang et al ., 2001). Therefore, by determining the effect of these antioxidants it should be possible to establish whether oxidative damage has a role in telomere shortening. It has been shown that selective targeting of a potent antioxidant to mitochondria is achieved by attaching the redox active moiety of ubiquinol to the decyltriphenylphosphonium (DPPT) cation, resulting in the mitochondria-specific antioxidant mitoQ [10-(6 ′ -ubiquinonyl) decyltriphenylphosphonium bromide] (Kelso et al ., 2001). Ubiquinol acts as an antioxidant by donating a hydrogen atom from one of its hydroxyl groups to a lipid peroxyl radical, which decreases lipid peroxidation within the mitochondrial inner membrane (Ingold et al ., 1993). MitoQ reduces oxidative damage and decreases ROS-induced apoptosis in short-term experiments (Kelso et al ., 2001; Echtay et al ., 2002). As mitoQ is predominantly located within mitochondria in cells due to its accumulation by the mitochondrial membrane potential, its effects in cells are thought to be largely due to the prevention of mitochondrial oxidative damage (Kelso et al ., 2001) and there is also evidence that mitoQ decreases the release of ROS from mitochondria (Hwang et al ., 2001). The possibility of such effects being due to non-specific interactions with mitochondria within cells can be discounted by the use of control compounds such as DPPT, which are also accumulated within mitochondria driven by the membrane potential but which do not act as antioxidants. Therefore, the blocking of a process by mitoQ but not by DPPT indicates a role for ROS production in the process and is consistent with the increased ROS production being primarily mitochondrial. Telomeres act as ‘mitotic clocks’ in human fibroblasts because they shorten with each round of replication due to both the inability of DNA polymerases to replicate the very ends of chromosomes (Olovnikow, 1973) and the specific accumulation of stress-induced DNA damage (von Zglinicki, 2002). Eventually, telomere dysfunction triggers replicative senescence (Bodnar et al ., 1998). Although intense stress can cause a senescencelike arrest without involvement of telomeres (Chen et al ., 2001; Gorbunova et al ., 2002), one possibility is that ROS production accelerates replicative senescence via its contribution to telomere shortening under conditions of mild stress. Therefore, we wanted to find out whether mitoQ could prolong the replicative lifespan of human fibroblasts under mild stress conditions, and whether this would correlate with a reduction in the rate of telomere shortening. MitoQ in micromolar concentrations selectively blocks mitochondrial oxidative damage and prevents apoptosis induced by acute treatments with hydrogen peroxide (Kelso et al ., 2001). When mitoQ was incubated with MRC-5 fibroblasts, concentrations above 50–100 n M were cytostatic in long-term culture, and even for concentrations of 10–20 n M an adaptation period of at least one week under normoxic conditions was necessary before beneficial effects on growth could be seen (data not shown). Such an adaptation period seems to be a characteristic effect of powerful antioxidants and may reflect the involvement of ROS in a multitude of cellular signal transduction chains (Forman et al ., 2002). Neither DPPT nor mitoQ had a significant effect on the intracellular peroxide content under normoxic culture conditions as measured by 2 ′ ,7 ′ -dichlorofluorescein fluorescence. However, mitoQ, but not DPPT, abolished nearly half of the rise in peroxides induced by hyperoxic culture in untreated cultures (Fig. 1a). Chronically increased oxidative stress exerted by culture under mild hyperoxia (40% oxygen partial pressure) shortens the replicative lifespan of MRC-5 fibroblasts down to few population doublings (von Zglinicki et al ., 1995; von Zglinicki, 2002). This premature aging phenotype is indistinguishable from replicative senescence under standard culture conditions (von Zglinicki et al ., 1995; Saretzki et al ., 1998; Toussaint et al ., 2000). Correspondence Professor T. von Zglinicki, Gerontology, Institute of Aging and Health, Newcastle University, General Hospital, Westgate Road, Newcastle upon Tyne NE4 6BE, UK. Tel.: 0191 2563310; fax: 0191 2195074; e-mail: t.vonzglinicki@ncl.ac.uk