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 (Fe(2+)) and hydrogen peroxide (H(2)O(2)) are indispensable for thyroid hormone synthesis, therefore being available in physiological conditions presumably at high concentrations. They participate in Fenton reaction (Fe(2+)+H(2)O(2)→Fe(3+)+(·)OH + OH(-)), resulting in the formation of the most harmful free radical – hydroxyl radical ((·)OH). 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 H(2)O(2) [100, 10, 1.0, 0.5, 0.1, 0.001, 0.00001 mM] or FeSO(4) (Fe(2+)) [300, 150, 30, 15, 3.0, 1.5 μM], or in the presence of those two factors used together, namely, in the presence of Fe(2+) [30 μM] plus H(2)O(2) [100, 10, 1.0, 0.5, 0.1, 0.001, 0.00001 mM], or in the presence of H(2)O(2) [0.5 mM] plus Fe(2+) [300, 150, 30, 15, 3.0, 1.5 μM]. 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 H(2)O(2) concentration of 100 mM and in Fe(2+) concentration-dependent manner [300, 150, and 30 μM]. When Fe(2+) and H(2)O(2) were applied together, Fe(2+) enhanced H(2)O(2) damaging effect to a higher degree than did H(2)O(2) on Fe(2+) 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.