High level of oxidized nucleosides in thyroid mitochondrial DNA; damaging effects of Fenton reaction substrates

Background: The mitochondrial DNA ( mtDNA) lies in close proximity to the free radical-producing electron transport chain, thus, it is highly prone to oxidative damage. Oxyphilic type of follicular thyroid carcinoma consists of cells filled - almost exclusively - with aberrant mitochondria. In turn, bivalent iron (Fe2+) and hydrogen peroxide (H2O2) are indispensable for thyroid hormone synthesis, therefore being available in physiological conditions presumably at high concentrations. They participate in Fenton reaction (Fe2++H2O2 -> Fe3++(OH)-O-center dot+OH-), resulting in the formation of the most harmful free radical - hydroxyl radical ((OH)-O-center dot). The same substrates may be used to experimentally induce oxidative damage to macromolecules. The aim of the study was to evaluate the background level of oxidative damage to mtDNA and the damaging effects of Fenton reaction substrates. Methods: Thyroid mtDNA was incubated in the presence of either H2O2 [100, 10, 1.0, 0.5, 0.1, 0.001, 0.00001 mM] or FeSO4 (Fe2+) [300, 150, 30, 15, 3.0, 1.5 mu M], or in the presence of those two factors used together, namely, in the presence of Fe2+ [30 mu M] plus H2O2 [100, 10, 1.0, 0.5, 0.1, 0.001, 0.00001 mM], or in the presence of H2O2 [0.5 mM] plus Fe2+[300, 150, 30, 15, 3.0, 1.5 mu M]. 8-oxo-7,8- dihydro-2'-deoxyguanosine (8-oxodG) concentration, as the index of DNA damage, was measured by HPLC. Results: Both Fenton reaction substrates, used separately, increased 8-oxodG level for the highest H2O2 concentration of 100 mM and in Fe2+ concentration-dependent manner [300, 150, and 30 mu M]. When Fe2+ and H2O2 were applied together, Fe2+ enhanced H2O2 damaging effect to a higher degree than did H2O2 on Fe2+ effect. Conclusions: The level of oxidized nucleosides in thyroid mtDNA is relatively high, when compared to nuclear DNA. Both substrates of Fenton reaction, i.e. ferrous ion and hydrogen peroxide, increase oxidative damage to mtDNA, with stronger damaging effect exerted by iron. High level of oxidative damage to mtDNA suggests its possible contribution to malignant transformation of thyroid oncocytic cells, which are known to be especially abundant in mitochondria, the latter characterized by molecular and enzymatic abnormalities.