Fernando Dutra

Oxidative damage to ferritin by 5-aminolevulinic acid.

5-Aminolevulinic acid (ALA), a heme precursor overproduced in various porphyric disorders, has been implicated in iron-mediated oxidative damage to biomolecules and cell structures. From previous observations of ferritin iron release by ALA, we investigated the ability of ALA to cause oxidative damage to ferritin apoprotein. Incubation of horse spleen ferritin (HoSF) with ALA caused alterations in the ferritin circular dichroism spectrum (loss of a alpha-helix content) and altered electrophoretic behavior. Incubation of human liver, spleen, and heart ferritins with ALA substantially decreased antibody recognition (51, 60, and 28% for liver, spleen, and heart, respectively). Incubation of apoferritin with 1-10mM ALA produced dose-dependent decreases in tryptophan fluorescence (11-35% after 5h), and a partial depletion of protein thiols (18% after 24h) despite substantial removal of catalytic iron. The loss of tryptophan fluorescence was inhibited 35% by 50mM mannitol, suggesting participation of hydroxyl radicals. The damage to apoferritin had no effect on ferroxidase activity, but produced a 61% decrease in iron uptake ability. The results suggest a local autocatalytic interaction among ALA, ferritin, and oxygen, catalyzed by endogenous iron and phosphate, that causes site-specific damage to the ferritin protein and impaired iron sequestration. These data together with previous findings that ALA overload causes iron mobilization in brain and liver of rats may help explain organ-specific toxicities and carcinogenicity of ALA in experimental animals and patients with porphyria.

Aminoacetone, a putative endogenous source of methylglyoxal, causes oxidative stress and death to insulin-producing RINm5f cells.

Aminoacetone (AA), triose phosphates, and acetone are putative endogenous sources of potentially cytotoxic and genotoxic methylglyoxal (MG), which has been reported to be augmented in the plasma of diabetic patients. In these patients, accumulation of MG derived from aminoacetone, a threonine and glycine catabolite, is inferred from the observed concomitant endothelial overexpression of circulating semicarbazide-sensitive amine oxidases. These copper-dependent enzymes catalyze the oxidation of primary amines, such as AA and methylamine, by molecular oxygen, to the corresponding aldehydes, NH4(+) ion and H2O2. We recently reported that AA aerobic oxidation to MG also takes place immediately upon addition of catalytic amounts of copper and iron ions. Taking into account that (i) MG and H2O2 are reportedly cytotoxic to insulin-producing cell lineages such as RINm5f and that (ii) the metal-catalyzed oxidation of AA is propagated by O2(*-) radical anion, we decided to investigate the possible pro-oxidant action of AA on these cells taken here as a reliable model system for pancreatic beta-cells. Indeed, we show that AA (0.10-5.0 mM) administration to RINm5f cultures induces cell death. Ferrous (50-300 microM) and Fe(3+) ion (100 microM) addition to the cell cultures had no effect, whereas Cu(2+) (5.0-100 microM) significantly increased cell death. Supplementation of the AA- and Cu(2+)-containing culture medium with antioxidants, such as catalase (5.0 microM), superoxide dismutase (SOD, 50 U/mL), and N-acetylcysteine (NAC, 5.0 mM) led to partial protection. mRNA expression of MnSOD, CuZnSOD, glutathione peroxidase, and glutathione reductase, but not of catalase, is higher in cells treated with AA (0.50-1.0 mM) plus Cu(2+) ions (10-50 microM) relative to control cultures. This may imply higher activity of antioxidant enzymes in RINm5f AA-treated cells. In addition, we have found that AA (0.50-1.0 mM) plus Cu(2+) (100 microM) (i) increase RINm5f cytosolic calcium; (ii) promote DNA fragmentation; and (iii) increase the pro-apoptotic (Bax)/antiapoptotic (Bcl-2) ratio at the level of mRNA expression. In conclusion, although both normal and pathological concentrations of AA are probably much lower than those used here, it is tempting to propose that excess AA in diabetic patients may drive oxidative damage and eventually the death of pancreatic beta-cells.

Aminoacetone Induces Loss of Ferritin Ferroxidase and Iron Uptake Activities

Aminoacetone (AA) is a threonine and glycine metabolite overproduced and recently implicated as a contributing source of methylglyoxal (MG) in conditions of ketosis. Oxidation of AA to MG, NH4+, and H2O2 has been reported to be catalyzed by a copper-dependent semicarbazide sensitive amine oxidase (SSAO) as well as by copper- and iron ion-catalyzed reactions with oxygen. We previously demonstrated that AA-generated O2*-. and enoyl radical (AA*) induce dose-dependent Fe(II) release from horse spleen ferritin (HoSF); no reaction occurs under nitrogen. In the present study we further explored the mechanism of iron release and the effect of AA on the ferritin apoprotein. Iron chelators such as EDTA, ATP and citrate, and phosphate accelerated AA-promoted iron release from HoSF, which was faster in horse spleen isoferritins containing larger amounts of phosphate in the core. Incubation of apoferritin with AA (2.5-50 mM, after 6 h) changes the apoprotein electrophoretic behavior, suggesting a structural modification of the apoprotein by AA-generated ROS. Superoxide dismutase (SOD) was able to partially protect apoferritin from structural modification whereas catalase, ethanol, and mannitol were ineffective in protection. Incubation of apoferritin with AA (1-10 mM) produced a dose-dependent decrease in tryptophan fluorescence (13-30%, after 5 h), and a partial depletion of protein thiols (29% after 24 h). The AA promoted damage to apoferritin produced a 40% decrease in apoprotein ferroxidase activity and an 80% decrease in its iron uptake ability. The current findings of changes in ferritin and apoferritin may contribute to intracellular iron-induced oxidative stress during AA formation in ketosis and diabetes mellitus.

Bioquímica e ação citotóxica de alfa-aminocetonas endógenas

a-Aminoketones are expected to undergo enolization and subsequent aerobic oxidation yielding oxyradicals and highly toxic a-oxoaldehydes. Our interest has been focused on two endogenous a-aminoketones: 5-aminolevulinic acid (ALA) and aminoacetone (AA), accumulated in porphyrias and diabetes mellitus, respectively, and recently implicated as contributing sources of oxyradicals in these diseases. The final oxidation product of ALA, 4,5-dioxovaleric acid (DOVA), is able to alkylate DNA guanine moieties and expected to promote protein cross-linking. Methylglyoxal (MG), the final oxidation product of AA, is also highly cytotoxic and able to aggregate protein molecules. This review covers chemical and biochemical aspects of these a-aminoketones and their putative roles in the oxidative stress associated with porphyric disorders and diabetes.

Liver Damage Induced by Succinylacetone: A Shared Redox Imbalance Mechanism between Tyrosinemia and Hepatic Porphyrias

To show liver failure mediated by 5-aminolevulinic acid (ALA), a heme precursor accumulated in inborn and acquired porphyrias, rats were treated with succinylacetone methyl ester (SAME). Treated rats underwent the expected ALA accumulation in plasma, liver and urine as a result from inhibition of ALA dehydratase (ALAD) activity. The enzyme activity decreased concomitantly with diminished urinary coproporphyrin levels. Additionally, liver protein carbonyls, iron and ferritin were higher in groups treated with a lower concentration of SAME whereas malondialdehyde was higher in the group treated with a higher ester dose. Consistent with these biochemical data, chronic treatment SAME was associated with induced oxidative subcellular and tissue damage revealed by cytosolic and mitochondrial changes within the liver cells. Altogether, these data expand the understanding of the direct biochemical mechanism for liver cell death in hepatic inborn disorders by generating excess ALA and may foster future therapeutic-driven strategies to preserve liver function.