Assessing the mechanism of iron toxicity in isolated liver mitochondria: oxidative damage and mitochondrial function

MUSACCO SEBIO, R; SAPORITO MAGRIÑÁ, C; BAJICOFF, S; PAREDES FLEITAS, P; ACOSTA, JM; BOVERIS, A; REPETTO, M

Buenos Aires

Congreso; 2nd International Congress in Translational Medicine; 2015

Iron (Fe) is a bioelement since Fe ions are involved in the basic biochemistry of aerobic life as part of the active centre of vital proteins and enzymes. However its deficiency or excess in the organism leads to pathologic situations. Fe overload and toxicity is associated to the generation of hydroxyl radical (HO.) from the reaction of Fe ions and hydrogen peroxide (H2O2) and superoxide anion (O2.-) by the Fenton/Haber-Weiss mechanism, leading to oxidative stress and damage to biomolecules. The aim of the work was to analyze the participation of O2.- and H2O2 in Fe toxicity in a biological environment. For that, an ex vivo model of isolated rat liver mitochondria was used. Oxidative damage to phospholipids was assessed by malondyaldehide content while mitochondrial function was assessed by respirometry in a Clark-type oxygen electrode. It was found that while very high iron concentrations (around 2 mM) were needed to affect mitochondrial function (oxygen consumption and respiratory control) in a situation of low O2.- and H2O2 production, much less metal concentration (0.5 mM) was needed to produce the same damage in a situation of high production of O2.- and H2O2. This situation was achieved by 10-minute incubation of mitochondria in presence of respiration substrates, which is known to produce a maximum in mitochondrial O2.- steady state concentration. Dose-dependent phospholipid oxidation was also found. This Fe-induced phospholipid oxidation in isolated mitochondria was found to occurr only when respiratory substrates are present in the respiratory medium, which means O2.- and H2O2 production. Only the lipophilic antioxidant butylated hydroxitoluene (BHT), which is a radical trapping agent, was able to prevent in a 100% the phospholipid oxidation as well as produced a slight improvement in the decrease in the respiratory function in mitochondria. The hydrophilic antioxidants glutathione (GSH) and N-acetylcysteine exhibited no effect in Fe-induced mitochondrial dysfunction and oxidative damage to phospholipids. The loss of mitochondrial function is related to the oxidative damage produced, leading to a disruption in the mitochondrial membrane structure where electron transfer chain and oxidative phosphorylation take place. These experiments showed the Fenton/Haber-Weiss reaction occurring in a biologic environment in a model of isolated rat liver mitochondria.