Abstract
Iron imbalance/accumulation has been implicated in oxidative injury associated with many degenerative diseases such as hereditary hemochromatosis, beta-thalassemia, and Friedreich's ataxia. Mitochondria are particularly sensitive to iron-induced oxidative stress - high loads of iron cause extensive lipid peroxidation and membrane permeabilization in isolated mitochondria. Here we detected and characterized mitochondrial DNA damage in isolated rat liver mitochondria exposed to a Fe2+-citrate complex, a small molecular weight complex. Intense DNA fragmentation was induced after the incubation of mitochondria with the iron complex. The detection of 3' phosphoglycolate ends at the mtDNA strand breaks by a 32P-postlabeling assay, suggested the involvement of hydroxyl radical in the DNA fragmentation induced by Fe2+-citrate. Increased levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine also suggested that Fe2+-citrate-induced oxidative stress causes mitochondrial DNA damage. In conclusion, our results show that iron-mediated lipid peroxidation was associated with intense mtDNA damage derived from the direct attack of reactive oxygen species.
Highlights
Iron is an essential element, necessary for the catalytic activity of a large number of enzymes
Southern hybridization of isolated liver mitochondrial DNA (mtDNA) with a HindIII fragment of mtDNA from C. hominivorax (Vargas and Azeredo-Espin 1995) revealed two bands in the lanes containing the samples extracted from rat liver mitochondria (RLM) (Fig. 1, lanes 1-3), corresponding to the supercoiled and closed circular forms of mtDNA, comparable to bands of mtDNA isolated from C. hominivorax
In samples obtained from control RLM, incubated with EGTA alone (Fig. 2B, lane 2), mtDNA was detected in its three archetypal conformations: supercoiled, open relaxed and linear
Summary
Iron is an essential element, necessary for the catalytic activity of a large number of enzymes. Mitochondria play a crucial role in iron homeostasis (Foury and Talibi 2001) and synthesis of ironsulfur clusters (Beinert et al 1997) and heme (Ryter and Tyrrell 2000). Unbalanced iron homeostasis can have severe consequences. It is well recognized that several physiological and pathological processes are linked to oxidative stress, defined as an imbalance between the rates of ROS formation and detoxification (Halliwell and Gutteridge 1999). In this regard, mitochondria are important cellular sites of ROS production and oxidative damage caused by these species (reviewed by Kowaltowski et al 2001)
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