Effect of Iron on Mitochondrial Function and RNA Oxidation

Objectives Iron is a redox reactive metal and causes reactive oxygen species formation via the Fenton reaction; Iron(II) is oxidized by hydrogen peroxide (H 2 O 2 ) to iron(III), thereby forming the reactive hydroxyl radical. Subsequently, the hydroxyl radical can cause oxidative damage to cellular macromolecules. The mitochondria contribute to H 2 O 2 formation and also take part in iron metabolism. We wanted to investigate if iron has an effect on mitochondrial function. Furthermore, we wanted to examine if iron increased RNA oxidation. Methods Experiments were performed on permeabilized mice muscle fibers. Mitochondrial function was measured by the Oxygraph-2k (Oroboros Instruments) as respiration and H 2 O 2 production. Iron (iron(II)sulfate, 0.1mM or 1 mM) was added during OXPHOS state (maximal respiration). Effect of iron chelator DFOM (0.1mM) was tested. In separate experiments permeabilized fibers were incubated for an hour with iron (0.01mM, 0.1mM and 1mM) and RNA oxidation was measured as 8-oxo-7,8-dihydroguanosine (8-oxoGuo) per 106 guanosine by UPLC-MS/MS. Results Addition of iron did not change the mitochondrial respiration (p=0.9). Addition of DFOM did not have an effect on respiration when comparing post-iron and post-DFOM addition (p=0.4). H 2 O 2 production increased only when 1mM iron was added (p=0.05), however this was also observed in experiments without a biopsy. DFOM decreased H 2 O 2 production in experiments with the biopsies, but had surprisingly no effect when no biopsy was present. We saw a significant 8-oxoGuo increase when adding iron, the increase being higher, the higher the iron concentration (p Conclusions Iron does not have an acute effect on mitochondrial OXPHOS state or H 2 O 2 production in mice biopsies. The observed increase in H 2 O 2 production is suspected to be caused by a chemical reaction between iron and the used buffer. However, addition of iron causes RNA oxidation. The acute effect of iron on RNA oxidation does not seem to be mediated by mitochondrial damage.