D-Amino acid containing peptides have been found to be responsible for saw¯y larvae poisoning in ... more D-Amino acid containing peptides have been found to be responsible for saw¯y larvae poisoning in many parts of the world. These compounds, unique in the animal kingdom, were isolated from three dierent species of saw¯y indigenous to Australia, Denmark and South America. The octapeptide, lophyrotomin, is the major toxin in the Australian and Danish species and is present in small amounts in the South American saw¯y. Pergidin, the main toxin in the South American saw¯y, is a heptapeptide containing a phosphoseryl residue. This, as far as we are aware, is the ®rst example of such a peptide to be isolated from an animal source. Small amounts of pergidin have been found in the other two species. All available evidence suggests that both peptides are biosynthesised`de novo' possibly as a protective device, however it cannot be excluded that microorganisms may be responsible. These compounds are stable to enzymatic breakdown because of their con®guration and their strong chemical bonding and lipophilic character provide a potential for residues to remain in the host animal and cause signi®cant changes. #
Aminoacetone (AA) is a threonine and glycine catabolite long known to accumulate in cridu-chat an... more Aminoacetone (AA) is a threonine and glycine catabolite long known to accumulate in cridu-chat and threoninemia syndromes and, more recently, implicated as a contributing source of methylglyoxal (MG) in diabetes mellitus. Oxidation of AA to MG, NH 4 + , and H 2 O 2 has been reported to be catalyzed by a copper-dependent semicarbazide sensitive amine oxidase (SSAO) as well as by Cu(II) ions. We here study the mechanism of AA aerobic oxidation, in the presence and absence of iron ions, and coupled to iron release from ferritin. Aminoacetone (1-7 mM) autoxidizes in Chelex-treated phosphate buffer (pH 7.4) to yield stoichiometric amounts of MG and NH 4 + . Superoxide radical was shown to propagate this reaction as indicated by strong inhibition of oxygen uptake by superoxide dismutase (SOD) (1-50 units/mL; up to 90%) or semicarbazide (0.5-5 mM; up to 80%) and by EPR spin trapping studies with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), which detected the formation of the DMPO-• OH adduct as a decomposition product from the DMPO-O 2 •adduct. Accordingly, oxygen uptake by AA is accelerated upon addition of xanthine/xanthine oxidase, a well-known enzymatic source of O 2 •radicals. Under Fe(II)EDTA catalysis, SOD (<50 units/mL) had little effect on the oxygen uptake curve or on the EPR spectrum of AA/DMPO, which shows intense signals of the DMPO-• OH adduct and of a secondary carbon-centered DMPO adduct, attributable to the AA • enoyl radical. In the presence of iron, simultaneous (two) electron transfer from both Fe(II) and AA to O 2 , leading directly to H 2 O 2 generation followed by the Fenton reaction is thought to take place. Aminoacetone was also found to induce dose-dependent Fe(II) release from horse spleen ferritin, putatively mediated by both O 2 •and AA • enoyl radicals, and the co-oxidation of added hemoglobin and myoglobin, which may be viewed as the initial step for potential further iron release. It is thus tempting to propose that AA, accumulated in the blood and other tissues of diabetics, besides being metabolized by SSAO, may release iron and undergo spontaneous and iron-catalyzed oxidation with production of reactive H 2 O 2 and O 2 •-, triggering pathological responses. It is noteworthy that noninsulin-dependent diabetes has been frequently associated with iron overload and oxidative stress.
Diacetyl, like other R-dicarbonyl compounds, is reportedly cytotoxic and genotoxic. A food and ci... more Diacetyl, like other R-dicarbonyl compounds, is reportedly cytotoxic and genotoxic. A food and cigarette contaminant, it is related with alcohol hepatotoxicity and lung disease. Peroxynitrite is a potent oxidant formed in vivo by the diffusion-controlled reaction of the superoxide radical anion with nitric oxide, which is able to form adducts with carbon dioxide and carbonyl compounds. Here, we investigate the nucleophilic addition of peroxynitrite to diacetyl forming acetyl radicals, whose reaction with molecular oxygen leads to acetate. Peroxynitrite is shown to react with diacetyl in phosphate buffer (bell-shaped pH profile with maximum at 7.2) at a very high rate constant (k 2 ) 1.0 × 10 4 M -1 s -1 ) when compared with monocarbonyl substrates (k 2 < 10 3 M -1 s -1 ). Phosphate ions (100-500 mM) do not affect the rate of spontaneous peroxynitrite decay, but the H 2 PO 4 anion catalyzes the nucleophilic addition of the peroxynitrite anion to diacetyl. The intermediacy of acetyl radicals is suggested by a three-line spectrum (a N ) a H ) 0.83 mT) obtained by EPR spin trapping of the reaction mixture with 2-methyl-2nitrosopropane. The peroxynitrite reaction is accompanied by concentration-dependent oxygen uptake. Stoichiometric amounts of acetate from millimolar amounts of peroxynitrite and diacetyl were obtained under nonlimiting conditions of dissolved oxygen. In the presence of either L-histidine or 2′deoxyguanosine, the peroxynitrite/diacetyl system afforded the corresponding acetylated molecules identified by HPLC-MS n . These studies provide evidence that the peroxynitrite/diacetyl reaction yields acetyl radicals and raise the hypothesis that protein and DNA nonenzymatic acetylation may occur in cells and be implicated in aging and metabolic disorders in which oxygen and nitrogen reactive species are putatively involved.
We describe herein the discovery of LASSBio-881 (3c) as a novel in vivo antinociceptive, anti-inf... more We describe herein the discovery of LASSBio-881 (3c) as a novel in vivo antinociceptive, anti-inflammatory, and in vitro antiproliferative and antioxidant compound, with a cannabinoid ligand profile. We observed that LASSBio-881 (3c) was able to bind to CB1 receptors (71% at 100 lM) and also to inhibit T-cell proliferation (66% at 10 lM) probably by binding to CB2 receptors, in a non-proapoptotic manner, different from anandamide (1). It was also demonstrated that LASSBio-881 (3c) had an important antioxidant profile toward free radicals (DPPH and hydroxyl), probably due to its particular redox behavior, which reflects the presence of both nitro and 3,5-di-tert-butyl-4-hydroxyphenyl sub-units, as demonstrated by cyclic voltammetry studies. In addition, we showed that these structural sub-units are essential for the observed pharmacological activity.
Aminoacetone (AA) is a threonine and glycine metabolite overproduced and recently implicated as a... more 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.
BACKGROUND: Hypothermia is a frequent event in severe acute pancreatitis (AP) and its real effect... more BACKGROUND: Hypothermia is a frequent event in severe acute pancreatitis (AP) and its real effects on the normal pancreas have not been well demonstrated. Moreover, neither have its effects on the outcome of acute pancreatitis been fully investigated. One hypothesis is that oxidative stress may be implicated in lesions caused or treated by hypothermia. AIM OF THE STUDY: To investigate the effect of hypothermia in cerulein-induced acute pancreatitis (CIAP) in rats and the role played by oxidative stress in this process. METHODS: Male Wistar rats were divided into hypothermic and normothermic groups. Hypothermia was induced with a cold mattress and rectal temperature was kept at 30ºC for one hour. Acute pancreatitis was induced with 2 doses of cerulein (20 ìg/kg) administered at a one-hour interval. Serum amylase, pancreas vascular permeability by Evan's blue method, pancreas wet-to-dry weight ratio and histopathology were analyzed in each group. RESULTS: When compared with normothermic rats, hypothermic animals, with cerulein-induced acute pancreatitis, showed higher levels of pancreatic vascular permeability (p < 0.05), pancreas wet-to-dry weight ratio (p = 0.03), and histologically verified edema (p < 0.05), but similar serum amylase levels. The hypothermic group showed a higher oxidized-reduced glutathione ratio than the normothermic group. CONCLUSION: Moderate hypothermia produced a greater inflammatory response in established acute pancreatitis induced by cerulein in rats. Moreover, this study suggests that oxidative stress may be one of the mechanisms responsible for the worse outcome in hypothermic rats with cerulein-induced acute pancreatitis.
Comparative Biochemistry and Physiology C-toxicology & Pharmacology, 2007
Amino metabolites with potential prooxidant properties, particularly α-aminocarbonyls, are the fo... more Amino metabolites with potential prooxidant properties, particularly α-aminocarbonyls, are the focus of this review. Among them we emphasize 5-aminolevulinic acid (a heme precursor formed from succinyl-CoA and glycine), aminoacetone (a threonine and glycine metabolite), and hexosamines and hexosimines, formed by Schiff condensation of hexoses with basic amino acid residues of proteins. All these metabolites were shown, in vitro, to undergo enolization and subsequent aerobic oxidation, yielding oxyradicals and highly cyto-and genotoxic α-oxoaldehydes. Their metabolic roles in health and disease are examined here and compared in humans and experimental animals, including rats, quail, and octopus. In the past two decades, we have concentrated on two endogenous α-aminoketones: (i) 5-aminolevulinic acid (ALA), accumulated in acquired (e.g., lead poisoning) and inborn (e.g., intermittent acute porphyria) porphyric disorders, and (ii) aminoacetone (AA), putatively overproduced in diabetes mellitus and cri-du-chat syndrome. ALA and AA have been implicated as contributing sources of oxyradicals and oxidative stress in these diseases. The end product of ALA oxidation, 4,5-dioxovaleric acid (DOVA), is able to alkylate DNA guanine moieties, promote protein cross-linking, and damage GABAergic receptors of rat brain synaptosome preparations. In turn, methylglyoxal (MG), the end product of AA oxidation, is also highly cytotoxic and able to release iron from ferritin and copper from ceruloplasmin, and to aggregate proteins. This review covers chemical and biochemical aspects of these α-aminoketones and their putative roles in the oxidative stress associated with porphyrias, tyrosinosis, diabetes, and cridu-chat. In addition, we comment briefly on a side prooxidant behaviour of hexosamines, that are known to constitute building blocks of several glycoproteins and to be involved in Schiff base-mediated enzymatic reactions.
a-Aminoketones are expected to undergo enolization and subsequent aerobic oxidation yielding oxyr... more 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.
Aminoacetone (AA), triose phosphates, and acetone are putative endogenous sources of potentially ... more 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, NH 4 + ion and H 2 O 2 . 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 H 2 O 2 are reportedly cytotoxic to insulin-producing cell lineages such as RINm5f and that (ii) the metal-catalyzed oxidation of AA is propagated by O 2
Aminoacetone (AA), a putative endogenous source of cytotoxic methylglyoxal, and ceruloplasmin (CP... more Aminoacetone (AA), a putative endogenous source of cytotoxic methylglyoxal, and ceruloplasmin (CP), the antioxidant plasma copper transporter, are known to increase in diabetes. AA was recently shown in vitro to act as a pro-oxidant toward ferritin and isolated mitochondria. We now report AA oxidative effects on CP mediated by AA-generated reactive oxygen species (ROS). Incubation of 1.5 µM human CP with 0.05-1 mM AA resulted in extensive protein aggregation. That ROS-driven thiol cross-linking underlies the CP aggregation was evidenced by the inhibitory effects of added superoxide dismutase, catalase, mannitol, and dithiothreitol. The addition of CP to AA (mM) solutions accelerated oxygen consumption by AA and caused CP copper ion release and loss of ferroxidase and aminoxidase activities. If operative in vivo, this reaction would impair the antioxidant role of CP and iron uptake by ferritin and hence contribute to intracellular iron-induced oxidative stress during AA accumulation in diabetes mellitus.
Aminoacetone (AA) is a threonine metabolite accumulated in threoninemia, cri-du-chat, and diabete... more Aminoacetone (AA) is a threonine metabolite accumulated in threoninemia, cri-du-chat, and diabetes, where it contributes toward the formation of cytotoxic and genotoxic methylglyoxal (MG). Oxyradicals yielded from iron-catalyzed AA aerobic oxidation to MG are shown here to promote Ca 2+ -mediated mitochondrial membrane permeabilization in an AA dose-dependent way. The inhibitory effect of added EGTA, cyclosporin A, Mg 2+ , and DTT observed in this study suggests the formation of transition pores in the inner mitochondrial membrane by AA, associated with thiol protein aggregation. That the mitochondrial iron pool plays a coadjutant role in the transition of mitochondrial permeability is indicated by the dramatic inhibitory effect of added o-phenanthroline. Iron released from ferritin by AA oxidation products-superoxide anion and AA enolyl radicals-is shown to act as an alternative source of ferrous iron, intensifying the mitochondrial damage. These findings may contribute to clarify the role of accumulated AA and iron overload in the mitochondrial oxidative damage reportedly occurring in diabetes mellitus.
5-Aminolevulinic acid (ALA), a heme precursor overproduced in various porphyric disorders, has be... more 5-Aminolevulinic acid (ALA), a heme precursor overproduced in various porphyric disorders, has been implicated in ironmediated 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 a-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 1-10 mM ALA produced dose-dependent decreases in tryptophan fluorescence (11-35% after 5 h), and a partial depletion of protein thiols (18% after 24 h) despite substantial removal of catalytic iron. The loss of tryptophan fluorescence was inhibited 35% by 50 mM 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.
Heme is an ancient and ubiquitous molecule present in organisms of all kingdoms, composed of an a... more Heme is an ancient and ubiquitous molecule present in organisms of all kingdoms, composed of an atom of iron linked to four ligand groups of porphyrin. A high amount of free heme, a potential amplifier of the inflammatory response, is a characteristic feature of diseases with increased hemolysis or extensive cell damage. Here we demonstrate that heme, but not its analogs/precursors, induced tumor necrosis factor-␣ (TNF-␣) secretion by macrophages dependently on MyD88, TLR4, and CD14. The activation of TLR4 by heme is exquisitely strict, requiring its coordinated iron and the vinyl groups of the porphyrin ring. Signaling of heme through TLR4 depended on an interaction distinct from the one established between TLR4 and lipopolysaccharide (LPS) since anti-TLR4/MD2 antibody or a lipid A antagonist inhibited LPS-induced TNF-␣ secretion but not heme activity. Conversely, protoporphyrin IX antagonized heme without affecting LPS-induced activation. Moreover, heme induced TNF-␣ and keratinocyte chemokine but was ineffective to induce interleukin-6, interleukin-12, and interferoninducible protein-10 secretion or co-stimulatory molecule expression. These findings support the concept that the broad ligand specificity of TLR4 and the different activation profiles might in part reside in its ability to recognize different ligands in different binding sites. Finally, heme induced oxidative burst, neutrophil recruitment, and heme oxygenase-1 expression independently of TLR4. Thus, our results presented here reveal a previous unrecognized role of heme as an extracellular signaling molecule that affects the innate immune response through a receptor-mediated mechanism.
D-Amino acid containing peptides have been found to be responsible for saw¯y larvae poisoning in ... more D-Amino acid containing peptides have been found to be responsible for saw¯y larvae poisoning in many parts of the world. These compounds, unique in the animal kingdom, were isolated from three dierent species of saw¯y indigenous to Australia, Denmark and South America. The octapeptide, lophyrotomin, is the major toxin in the Australian and Danish species and is present in small amounts in the South American saw¯y. Pergidin, the main toxin in the South American saw¯y, is a heptapeptide containing a phosphoseryl residue. This, as far as we are aware, is the ®rst example of such a peptide to be isolated from an animal source. Small amounts of pergidin have been found in the other two species. All available evidence suggests that both peptides are biosynthesised`de novo' possibly as a protective device, however it cannot be excluded that microorganisms may be responsible. These compounds are stable to enzymatic breakdown because of their con®guration and their strong chemical bonding and lipophilic character provide a potential for residues to remain in the host animal and cause signi®cant changes. #
Aminoacetone (AA) is a threonine and glycine catabolite long known to accumulate in cridu-chat an... more Aminoacetone (AA) is a threonine and glycine catabolite long known to accumulate in cridu-chat and threoninemia syndromes and, more recently, implicated as a contributing source of methylglyoxal (MG) in diabetes mellitus. Oxidation of AA to MG, NH 4 + , and H 2 O 2 has been reported to be catalyzed by a copper-dependent semicarbazide sensitive amine oxidase (SSAO) as well as by Cu(II) ions. We here study the mechanism of AA aerobic oxidation, in the presence and absence of iron ions, and coupled to iron release from ferritin. Aminoacetone (1-7 mM) autoxidizes in Chelex-treated phosphate buffer (pH 7.4) to yield stoichiometric amounts of MG and NH 4 + . Superoxide radical was shown to propagate this reaction as indicated by strong inhibition of oxygen uptake by superoxide dismutase (SOD) (1-50 units/mL; up to 90%) or semicarbazide (0.5-5 mM; up to 80%) and by EPR spin trapping studies with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), which detected the formation of the DMPO-• OH adduct as a decomposition product from the DMPO-O 2 •adduct. Accordingly, oxygen uptake by AA is accelerated upon addition of xanthine/xanthine oxidase, a well-known enzymatic source of O 2 •radicals. Under Fe(II)EDTA catalysis, SOD (<50 units/mL) had little effect on the oxygen uptake curve or on the EPR spectrum of AA/DMPO, which shows intense signals of the DMPO-• OH adduct and of a secondary carbon-centered DMPO adduct, attributable to the AA • enoyl radical. In the presence of iron, simultaneous (two) electron transfer from both Fe(II) and AA to O 2 , leading directly to H 2 O 2 generation followed by the Fenton reaction is thought to take place. Aminoacetone was also found to induce dose-dependent Fe(II) release from horse spleen ferritin, putatively mediated by both O 2 •and AA • enoyl radicals, and the co-oxidation of added hemoglobin and myoglobin, which may be viewed as the initial step for potential further iron release. It is thus tempting to propose that AA, accumulated in the blood and other tissues of diabetics, besides being metabolized by SSAO, may release iron and undergo spontaneous and iron-catalyzed oxidation with production of reactive H 2 O 2 and O 2 •-, triggering pathological responses. It is noteworthy that noninsulin-dependent diabetes has been frequently associated with iron overload and oxidative stress.
Diacetyl, like other R-dicarbonyl compounds, is reportedly cytotoxic and genotoxic. A food and ci... more Diacetyl, like other R-dicarbonyl compounds, is reportedly cytotoxic and genotoxic. A food and cigarette contaminant, it is related with alcohol hepatotoxicity and lung disease. Peroxynitrite is a potent oxidant formed in vivo by the diffusion-controlled reaction of the superoxide radical anion with nitric oxide, which is able to form adducts with carbon dioxide and carbonyl compounds. Here, we investigate the nucleophilic addition of peroxynitrite to diacetyl forming acetyl radicals, whose reaction with molecular oxygen leads to acetate. Peroxynitrite is shown to react with diacetyl in phosphate buffer (bell-shaped pH profile with maximum at 7.2) at a very high rate constant (k 2 ) 1.0 × 10 4 M -1 s -1 ) when compared with monocarbonyl substrates (k 2 < 10 3 M -1 s -1 ). Phosphate ions (100-500 mM) do not affect the rate of spontaneous peroxynitrite decay, but the H 2 PO 4 anion catalyzes the nucleophilic addition of the peroxynitrite anion to diacetyl. The intermediacy of acetyl radicals is suggested by a three-line spectrum (a N ) a H ) 0.83 mT) obtained by EPR spin trapping of the reaction mixture with 2-methyl-2nitrosopropane. The peroxynitrite reaction is accompanied by concentration-dependent oxygen uptake. Stoichiometric amounts of acetate from millimolar amounts of peroxynitrite and diacetyl were obtained under nonlimiting conditions of dissolved oxygen. In the presence of either L-histidine or 2′deoxyguanosine, the peroxynitrite/diacetyl system afforded the corresponding acetylated molecules identified by HPLC-MS n . These studies provide evidence that the peroxynitrite/diacetyl reaction yields acetyl radicals and raise the hypothesis that protein and DNA nonenzymatic acetylation may occur in cells and be implicated in aging and metabolic disorders in which oxygen and nitrogen reactive species are putatively involved.
We describe herein the discovery of LASSBio-881 (3c) as a novel in vivo antinociceptive, anti-inf... more We describe herein the discovery of LASSBio-881 (3c) as a novel in vivo antinociceptive, anti-inflammatory, and in vitro antiproliferative and antioxidant compound, with a cannabinoid ligand profile. We observed that LASSBio-881 (3c) was able to bind to CB1 receptors (71% at 100 lM) and also to inhibit T-cell proliferation (66% at 10 lM) probably by binding to CB2 receptors, in a non-proapoptotic manner, different from anandamide (1). It was also demonstrated that LASSBio-881 (3c) had an important antioxidant profile toward free radicals (DPPH and hydroxyl), probably due to its particular redox behavior, which reflects the presence of both nitro and 3,5-di-tert-butyl-4-hydroxyphenyl sub-units, as demonstrated by cyclic voltammetry studies. In addition, we showed that these structural sub-units are essential for the observed pharmacological activity.
Aminoacetone (AA) is a threonine and glycine metabolite overproduced and recently implicated as a... more 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.
BACKGROUND: Hypothermia is a frequent event in severe acute pancreatitis (AP) and its real effect... more BACKGROUND: Hypothermia is a frequent event in severe acute pancreatitis (AP) and its real effects on the normal pancreas have not been well demonstrated. Moreover, neither have its effects on the outcome of acute pancreatitis been fully investigated. One hypothesis is that oxidative stress may be implicated in lesions caused or treated by hypothermia. AIM OF THE STUDY: To investigate the effect of hypothermia in cerulein-induced acute pancreatitis (CIAP) in rats and the role played by oxidative stress in this process. METHODS: Male Wistar rats were divided into hypothermic and normothermic groups. Hypothermia was induced with a cold mattress and rectal temperature was kept at 30ºC for one hour. Acute pancreatitis was induced with 2 doses of cerulein (20 ìg/kg) administered at a one-hour interval. Serum amylase, pancreas vascular permeability by Evan's blue method, pancreas wet-to-dry weight ratio and histopathology were analyzed in each group. RESULTS: When compared with normothermic rats, hypothermic animals, with cerulein-induced acute pancreatitis, showed higher levels of pancreatic vascular permeability (p < 0.05), pancreas wet-to-dry weight ratio (p = 0.03), and histologically verified edema (p < 0.05), but similar serum amylase levels. The hypothermic group showed a higher oxidized-reduced glutathione ratio than the normothermic group. CONCLUSION: Moderate hypothermia produced a greater inflammatory response in established acute pancreatitis induced by cerulein in rats. Moreover, this study suggests that oxidative stress may be one of the mechanisms responsible for the worse outcome in hypothermic rats with cerulein-induced acute pancreatitis.
Comparative Biochemistry and Physiology C-toxicology & Pharmacology, 2007
Amino metabolites with potential prooxidant properties, particularly α-aminocarbonyls, are the fo... more Amino metabolites with potential prooxidant properties, particularly α-aminocarbonyls, are the focus of this review. Among them we emphasize 5-aminolevulinic acid (a heme precursor formed from succinyl-CoA and glycine), aminoacetone (a threonine and glycine metabolite), and hexosamines and hexosimines, formed by Schiff condensation of hexoses with basic amino acid residues of proteins. All these metabolites were shown, in vitro, to undergo enolization and subsequent aerobic oxidation, yielding oxyradicals and highly cyto-and genotoxic α-oxoaldehydes. Their metabolic roles in health and disease are examined here and compared in humans and experimental animals, including rats, quail, and octopus. In the past two decades, we have concentrated on two endogenous α-aminoketones: (i) 5-aminolevulinic acid (ALA), accumulated in acquired (e.g., lead poisoning) and inborn (e.g., intermittent acute porphyria) porphyric disorders, and (ii) aminoacetone (AA), putatively overproduced in diabetes mellitus and cri-du-chat syndrome. ALA and AA have been implicated as contributing sources of oxyradicals and oxidative stress in these diseases. The end product of ALA oxidation, 4,5-dioxovaleric acid (DOVA), is able to alkylate DNA guanine moieties, promote protein cross-linking, and damage GABAergic receptors of rat brain synaptosome preparations. In turn, methylglyoxal (MG), the end product of AA oxidation, is also highly cytotoxic and able to release iron from ferritin and copper from ceruloplasmin, and to aggregate proteins. This review covers chemical and biochemical aspects of these α-aminoketones and their putative roles in the oxidative stress associated with porphyrias, tyrosinosis, diabetes, and cridu-chat. In addition, we comment briefly on a side prooxidant behaviour of hexosamines, that are known to constitute building blocks of several glycoproteins and to be involved in Schiff base-mediated enzymatic reactions.
a-Aminoketones are expected to undergo enolization and subsequent aerobic oxidation yielding oxyr... more 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.
Aminoacetone (AA), triose phosphates, and acetone are putative endogenous sources of potentially ... more 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, NH 4 + ion and H 2 O 2 . 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 H 2 O 2 are reportedly cytotoxic to insulin-producing cell lineages such as RINm5f and that (ii) the metal-catalyzed oxidation of AA is propagated by O 2
Aminoacetone (AA), a putative endogenous source of cytotoxic methylglyoxal, and ceruloplasmin (CP... more Aminoacetone (AA), a putative endogenous source of cytotoxic methylglyoxal, and ceruloplasmin (CP), the antioxidant plasma copper transporter, are known to increase in diabetes. AA was recently shown in vitro to act as a pro-oxidant toward ferritin and isolated mitochondria. We now report AA oxidative effects on CP mediated by AA-generated reactive oxygen species (ROS). Incubation of 1.5 µM human CP with 0.05-1 mM AA resulted in extensive protein aggregation. That ROS-driven thiol cross-linking underlies the CP aggregation was evidenced by the inhibitory effects of added superoxide dismutase, catalase, mannitol, and dithiothreitol. The addition of CP to AA (mM) solutions accelerated oxygen consumption by AA and caused CP copper ion release and loss of ferroxidase and aminoxidase activities. If operative in vivo, this reaction would impair the antioxidant role of CP and iron uptake by ferritin and hence contribute to intracellular iron-induced oxidative stress during AA accumulation in diabetes mellitus.
Aminoacetone (AA) is a threonine metabolite accumulated in threoninemia, cri-du-chat, and diabete... more Aminoacetone (AA) is a threonine metabolite accumulated in threoninemia, cri-du-chat, and diabetes, where it contributes toward the formation of cytotoxic and genotoxic methylglyoxal (MG). Oxyradicals yielded from iron-catalyzed AA aerobic oxidation to MG are shown here to promote Ca 2+ -mediated mitochondrial membrane permeabilization in an AA dose-dependent way. The inhibitory effect of added EGTA, cyclosporin A, Mg 2+ , and DTT observed in this study suggests the formation of transition pores in the inner mitochondrial membrane by AA, associated with thiol protein aggregation. That the mitochondrial iron pool plays a coadjutant role in the transition of mitochondrial permeability is indicated by the dramatic inhibitory effect of added o-phenanthroline. Iron released from ferritin by AA oxidation products-superoxide anion and AA enolyl radicals-is shown to act as an alternative source of ferrous iron, intensifying the mitochondrial damage. These findings may contribute to clarify the role of accumulated AA and iron overload in the mitochondrial oxidative damage reportedly occurring in diabetes mellitus.
5-Aminolevulinic acid (ALA), a heme precursor overproduced in various porphyric disorders, has be... more 5-Aminolevulinic acid (ALA), a heme precursor overproduced in various porphyric disorders, has been implicated in ironmediated 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 a-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 1-10 mM ALA produced dose-dependent decreases in tryptophan fluorescence (11-35% after 5 h), and a partial depletion of protein thiols (18% after 24 h) despite substantial removal of catalytic iron. The loss of tryptophan fluorescence was inhibited 35% by 50 mM 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.
Heme is an ancient and ubiquitous molecule present in organisms of all kingdoms, composed of an a... more Heme is an ancient and ubiquitous molecule present in organisms of all kingdoms, composed of an atom of iron linked to four ligand groups of porphyrin. A high amount of free heme, a potential amplifier of the inflammatory response, is a characteristic feature of diseases with increased hemolysis or extensive cell damage. Here we demonstrate that heme, but not its analogs/precursors, induced tumor necrosis factor-␣ (TNF-␣) secretion by macrophages dependently on MyD88, TLR4, and CD14. The activation of TLR4 by heme is exquisitely strict, requiring its coordinated iron and the vinyl groups of the porphyrin ring. Signaling of heme through TLR4 depended on an interaction distinct from the one established between TLR4 and lipopolysaccharide (LPS) since anti-TLR4/MD2 antibody or a lipid A antagonist inhibited LPS-induced TNF-␣ secretion but not heme activity. Conversely, protoporphyrin IX antagonized heme without affecting LPS-induced activation. Moreover, heme induced TNF-␣ and keratinocyte chemokine but was ineffective to induce interleukin-6, interleukin-12, and interferoninducible protein-10 secretion or co-stimulatory molecule expression. These findings support the concept that the broad ligand specificity of TLR4 and the different activation profiles might in part reside in its ability to recognize different ligands in different binding sites. Finally, heme induced oxidative burst, neutrophil recruitment, and heme oxygenase-1 expression independently of TLR4. Thus, our results presented here reveal a previous unrecognized role of heme as an extracellular signaling molecule that affects the innate immune response through a receptor-mediated mechanism.
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Papers by Fernando Dutra