Free radical generation during delta-aminolevulinic acid autoxidation: induction by hemoglobin and connections with porphyrinpathies

delta-Aminolevulinic acid (ALA), a heme precursor accumulated in acute intermittent porphyria and saturnism, undergoes autoxidation leading to ammonium ion and probably the corresponding alpha-ketoaldehyde. This reaction is accelerated by addition of oxyhemoglobin (oxyHb) and other iron complexes. OxyHb is concomitantly oxidized to metHb; the apparent second-order rate constant of oxyHb/ALA coupled oxidation is ca. 10 M-1 min-1.1H NMR and uv spectral studies suggest that ALA undergoes enolization before consuming the dissolved oxygen. Spin-trapping experiments demonstrate formation of both the hydroxyl radical and a substrate-derived carbon-centered radical during ALA oxidation. Generation of active oxygen species by ALA might be related to the neuropathy associated to some acquired and inherited porphyrinpathies.     

Iron autoxidation and free radical generation: effects of buffers, ligands, and chelators.

The pH of the solution along with chelation and consequently coordination of iron regulate its reactivity. In this study we confirmed that, in general, the rate of Fe(II) autoxidation increases as the pH of the solution is increased, but chelators that provide oxygen ligands for the iron can override the affect of pH. Additionally, the stoichiometry of the Fe(II) autoxidation reaction varied from 2:1 to 4:1, dependent upon the rate of Fe(II) autoxidation, which is dependent upon the chelator. No partially reduced oxygen species were detected during the autoxidation of Fe(II) by ESR using DMPO as the spin trap. However, upon the addition of ethanol to the assay, the DMPO:hydroxyethyl radical adduct was detected. Additionally, the hydroxylation of terephthalic acid by various iron-chelator complexes during the autoxidation of Fe(II) was assessed by fluorometric techniques. The oxidant formed during the autoxidation of EDTA:Fe(II) was shown to have different reactivity than the hydroxyl radical, suggesting that some type of hypervalent iron complex was formed. Ferrous iron was shown to be able to directly reduce some quinones without the reduction of oxygen. In conclusion, this study demonstrates the complexity of iron chemistry, especially the chelation of iron and its subsequent reactivity.     

Hypoxic reperfusion of the ischemic heart and oxygen radical generation

Postischemic myocardial contractile dysfunction is in part mediated by the burst of reactive oxygen species (ROS), which occurs with the reintroduction of oxygen. We hypothesized that tissue oxygen tension modulates this ROS burst at reperfusion. After 20 min of global ischemia, isolated rat hearts were reperfused with temperature-controlled (37.4 degrees C) Krebs-Henseleit buffer saturated with one of three different O2 concentrations (95, 20, or 2%) for the first 5 min of reperfusion and then changed to 95% O2. Additional hearts were loaded with 1) allopurinol (1 mM), a xanthine oxidase inhibitor, 2) diphenyleneiodonium (DPI; 1 microM), an NAD(P)H oxidase inhibitor, or 3) Tiron (10 mM), a superoxide scavenger, and were then reperfused with either 95 or 2% O2 for the first 5 min. ROS production and tissue oxygen tension were quantitated using electron paramagnetic resonance spectroscopy. Tissue oxygen tension was significantly higher in the 95% O2 group. However, the largest radical burst occurred in the 2% O2 reperfusion group (P < 0.001). Recovery of left ventricular (LV) contractile function and aconitase activity during reperfusion were inversely related to the burst of radical production and were significantly higher in hearts initially reperfused with 95% O2 (P < 0.001). Allopurinol, DPI, and Tiron reduced the burst of radical formation in the 2% O2 reperfusion groups (P < 0.05). Hypoxic reperfusion generates an increased ROS burst originating from multiple pathways. Recovery of LV function during reperfusion is inversely related to this oxygen radical burst, highlighting the importance of myocardial oxygen tension during initial reperfusion.     

Superoxide radical initiates the autoxidation of dihydroxyacetone

The aerobic xanthine oxidase reaction causes the cooxidation of dihydroxyacetone in a process which is strongly inhibited by superoxide dismutase but not by catalase, HO X scavengers, or iron-inactivating chelating agents. Several molecules of the sugar can be oxidized per O2- introduced. A free radical chain mechanism, in which O2- acts both as an initiator and as a chain propagator, is proposed. Simple sugars capable of tautomerizing to enediols may now be added to the list of biologically relevant targets for O2-.     

Response of mitochondrial reactive oxygen species generation to steady-state oxygen tension: implications for hypoxic cell signaling

Mitochondria are proposed to play an important role in hypoxic cell signaling. One currently accepted signaling paradigm is that the mitochondrial generation of reactive oxygen species (ROS) increases in hypoxia. This is paradoxical, because oxygen is a substrate for ROS generation. Although the response of isolated mitochondrial ROS generation to [O(2)] has been examined previously, such investigations did not apply rigorous control over [O(2)] within the hypoxic signaling range. With the use of open-flow respirometry and fluorimetry, the current study determined the response of isolated rat liver mitochondrial ROS generation to defined steady-state [O(2)] as low as 0.1 microM. In mitochondria respiring under state 4 (quiescent) or state 3 (ATP turnover) conditions, decreased ROS generation was always observed at low [O(2)]. It is concluded that the biochemical mechanism to facilitate increased ROS generation in response to hypoxia in cells is not intrinsic to the mitochondrial respiratory chain alone but may involve other factors. The implications for hypoxic cell signaling are discussed.     

Oxygen radical generation by microsomes: role of iron and implications for alcohol metabolism and toxicity

Experiments were carried out to evaluate whether the molecular mechanism for ethanol oxidation by microsomes, a minor pathway of alcohol metabolism, involved generation of hydroxyl radical (.OH). Microsomes oxidized chemical .OH scavengers (KMB, DMSO, t-butyl alcohol, benzoate) by a reaction sensitive to catalase, but not SOD. Iron was required for microsomal .OH generation in view of the potent inhibition by desferrioxamine; however, the chelated form of iron was important. Microsomal .OH production was effectively stimulated by ferric EDTA or ferric DTPA, but poorly increased with ferric ATP, ferric citrate, or ferric ammonium sulfate. By contrast, the latter ferric complexes effectively increased microsomal chemiluminescence and lipid peroxidation, whereas ferric EDTA and ferric DTPA were inhibitory. Under conditions that minimize .OH production (absence of EDTA, iron) ethanol was oxidized by a cytochrome P-450-dependent process independent of reactive oxygen intermediates. Under conditions that promote microsomal .OH production, the oxidation of ethanol by .OH becomes more significant in contributing to the overall oxidation of ethanol by microsomes. Experiments with inhibitors and reconstituted systems containing P-450 and NADPH-P-450 reductase indicated that the reductase is the critical enzyme locus for interacting with iron and catalyzing production of reactive oxygen species. Microsomes isolated from rats chronically fed ethanol catalyzed oxidation of .OH scavengers, light emission, and inactivation of added metabolic enzymes at elevated rates, and displayed an increase in ethanol oxidation by a .OH-dependent and a P-450-dependent pathway. It is possible that enhanced generation of reactive oxygen intermediates by microsomes may contribute to the hepatotoxic effects of ethanol.     

Phospholipid polar heads affect the generation of oxygen active species by Fe2+ autoxidation.

The possibility that phospholipid polar heads may influence Fe2+ reaction with molecular oxygen and, thus, the generation of oxygen active species was investigated. Dipalmitoyl phosphatidylcholine (DPPC) and DPPC/dipalmitoyl phosphatidic acid (DPPA) were utilized as model liposomes. Fe2+ oxidation, oxygen consumption, nitro blue tetrazolium reduction and 2-deoxyribose degradation were the parameters evaluated. Comparison of the results obtained clearly shows that the two types of polar heads differently affect iron chemistry. DPPC liposomes are ineffective. By contrast, Fe2+ oxidation by oxygen occurs in the presence of DPPC/DPPA liposomes. During this reaction, species able to reduce nitro blue tetrazolium and to degrade 2-deoxyribose are generated. The results obtained indicate that the polar heads of phospholipids, by influencing Fe2+ autoxidation, generate dangerous oxygen species which may exert an active role in the oxidation of the associated hydrophobic components of the phospholipids.     

Homogentisic acid induces aggregation and fibrillation of amyloidogenic proteins

BackgroundAlkaptonuria (AKU) is an ultra-rare inborn error of metabolism characterized by homogentisic acid (HGA) accumulation due to a deficient activity of the homogentisate 1.2-dioxygenase (HGD) enzyme. This leads to the production of dark pigments that are deposited onto connective tissues, a condition named ‘ochronosis’ and whose mechanisms are not completely clear. Recently, the potential role of hitherto unidentified proteins in the ochronotic process was hypothesized, and the presence of Serum Amyloid A (SAA) in alkaptonuric tissues was reported, allowing the classification of AKU as a novel secondary amyloidosis.MethodsGel electrophoresis, Western Blot, Congo Red-based assays and electron microscopy were used to investigate the effects of HGA on the aggregation and fibrillation propensity of amyloidogenic proteins and peptides [Aβ(1–42), transthyretin, atrial natriuretic peptide, α-synuclein and SAA]. LC/MS and in silico analyses were undertaken to identify possible binding sites for HGA (or its oxidative metabolite, a benzoquinone acetate or BQA) in SAA.ResultsWe found that HGA might act as an amyloid aggregation enhancer in vitro for all the tested proteins and peptides in a time- and dose- dependent fashion, and identified a small crevice at the interface between two HGD subunits as a candidate binding site for HGA/BQA.ConclusionsHGA might be an important amyloid co- component playing significant roles in AKU amyloidosis.General significanceOur results provide a possible explanation for the clinically verified onset of amyloidotic processes in AKU and might lay the basis to setup proper pharmacological approaches to alkaptonuric ochronosis, which are still lacking.     

Evidence for superoxide generation from the autoxidation of the favism-inducing aglycone divicine

The formation of the superoxide anion radical (O2-) during the autoxidation of divicine, an unstable aglycone involved in the hemolytic anemia occurring in favism, has been demonstrated by EPR with two different procedures. In the first case (chemical method) an O2--mediated reduction of a nitroxide by cysteine was shown to occur when divicine was allowed to cycle between the oxidized and the reduced form. In the second case (enzymatic method) the specific reaction between superoxide and superoxide dismutase was used as superoxide detector. It was shown that the enzyme attained a steady-state condition when mixed with divicine in the presence of air, as monitored by EPR evaluation of the oxidation state of the catalytic copper: this result is a direct, specific indicator of an O2- flux.     

Curcumin-induced inhibition of cellular reactive oxygen species generation: Novel therapeutic implications

There is evidence for increased levels of circulating reactive oxygen species (ROS) in diabetics, as indirectly inferred by the findings of increased lipid peroxidation and decreased antioxidant status. Direct measurements of intracellular generation of ROS using fluorescent dyes also demonstrate an association of oxidative stress with diabetes. Although phenolic compounds attenuate oxidative stress-related tissue damage, there are concerns over toxicity of synthetic phenolic antioxidants and this has considerably stimulated interest in investigating the role of natural phenolics in medicinal applications. Curcumin (the primary active principle in turmeric,Curcuma longa Linn.) has been claimed to represent a potential antioxidant and antiinflammatory agent with phytonutrient and bioprotective properties. However there are lack of molecular studies to demonstrate its cellular action and potential molecular targets. In this study the antioxidant effect of curcumin as a function of changes in cellular ROS generation was tested. Our results clearly demonstrate that curcumin abolished both phorbol-12 myristate-13 acetate (PMA) and thapsigargin-induced ROS generation in cells from control and diabetic subjects. The pattern of these ROS inhibitory effects as a function of dose-dependency suggests that curcumin mechanistically interferes with protein kinase C (PKC) and calcium regulation. Simultaneous measurements of ROS and Ca²⁺ influx suggest that a rise in cytosolic Ca²⁺ may be a trigger for increased ROS generation. We suggest that the antioxidant and antiangeogenic actions of curcumin, as a mechanism of inhibition of Ca²⁺ entry and PKC activity, should be further exploited to develop suitable and novel drugs for the treatment of diabetic retinopathy and other diabetic complications.     

Lipopolysaccharide-induced alterations in oxygen consumption and radical generation in endothelial cells

Oxygen consumption rate (OCR) and generation of superoxide and nitric oxide (NO) in mouse aortic endothelial cells (MAECs) treated with lipopolysaccharide (LPS) were studied. The OCR was determined in cell suspensions at 37 °C by electron paramagnetic resonance (EPR) spectroscopy. LPS significantly altered the OCR in a dose and time-dependent fashion. The OCR was significantly elevated immediately following the treatment of MAECs with LPS (5 and 10 μg/ml) and NADPH (100 μM) whereas the same was depressed 1 h after exposure to similar conditions of incubation. Under similar experimental conditions, superoxide generation was also determined by EPR spectroscopy and cytochrome c reduction assays. A marginal increase in the superoxide production was observed when the cells were treated with LPS and NADPH alone whereas the same was further enhanced significantly when the cells were treated with LPS and NADPH together. The increase in oxygen consumption and superoxide production caused by LPS was inhibited by diphenyleneiodonium (DPI), suggesting the involvement of NAD(P)H oxidase. A significant increase in the NO production by MAECs was noticed 1 h after treatment with LPS and was inhibited by L-NAME, further suggesting the involvement of nitric oxide synthase (NOS). Thus, on a temporal scale, LPS-induced alterations in oxygen consumption by MAECs may be under the control of dual regulation by NAD(P)H oxidase and NOS. (Mol Cell Biochem 278: 119–127, 2005)     

Generation of the superoxide radical during autoxidation of oxymyoglobin.

Autoxidation of bovine oxymyoglobin to metmyoglobin induces co-oxidation of epinephrine to adrenochrome. This co-oxidation is markedly inhibited by superoxide dismutase [EC 1.15.1.1]. Electron transfer from oxymyoglobin to ferricytochrome c is partially inhibited by superoxide dismutase. These results indicate that autoxidation of oxymyoglobin results in generation of superoxide radicals. Autoxidation of oxymyoglobin is accelerated by superoxide dismutase and partially inhibited by catalase [EC 1.11.1.6].     

Homogentisic acid induces morphological and mechanical aberration of ochronotic cartilage in alkaptonuria

Alkaptonuria (AKU) is a disease caused by a deficient homogentisate 1,2-dioxygenase activity leading to systemic accumulation of homogentisic acid (HGA), that forms a melanin-like polymer that progressively deposits onto connective tissues causing a pigmentation called “ochronosis” and tissue degeneration. The effects of AKU and ochronotic pigment on the biomechanical properties of articular cartilage need further investigation. To this aim, AKU cartilage was studied using thermal (thermogravimetry and differential scanning calorimetry) and rheological analysis. We found that AKU cartilage had a doubled mesopore radius compared to healthy cartilage. Since the mesoporous structure is the main responsible for maintaining a correct hydrostatic pressure and tissue homoeostasis, drastic changes of thermal and rheological parameters were found in AKU. In particular, AKU tissue lost its capability to enhance chondrocytes metabolism (decreased heat capacity) and hence the production of proteoglycans. A drastic increase in stiffness and decrease in dissipative and lubricant role ensued in AKU cartilage. Multiphoton and scanning electron microscopies revealed destruction of cell–matrix microstructure and disruption of the superficial layer. Such observations on AKU specimens were confirmed in HGA-treated healthy cartilage, indicating that HGA is the toxic responsible of morphological and mechanical alterations of cartilage in AKU.     

Steady-state kinetics of autoxidation of NAD(P)H initiated by hydroperoxyl radical, the acid form of superoxide anion radical.

Rates of autoxidation of NAD(P)H initiated by hydroperoxyl radical, the acid form of superoxide anion radical which was generated by xanthine/xanthine oxidase, followed a typical autoxidation kinetic equation. Second-order rate constants for the reactions of NADPH and NADH with hydroperoxyl radical were found to be 9.82 +/- 0.13 x 10(4) M-1s-1 and 9.26 +/- 0.58 x 10(4) M-1s-1 at 25 degrees C, respectively. Rates of the reactions between NAD(P)H and superoxide to give degraded products other than NAD(P)+ were also investigated.     

Paraquat- and diquat-induced oxygen radical generation and lipid peroxidation in rat brain microsomes

NADPH-menadione reductase activity by rat brain microsomes (Ms) was decreased 40-50% by 10 microM dicumarol, a potent inhibitor of DT-diaphorase, whereas no change in NADPH-paraquat (PQ) and -diquat (DQ) reductase activity was observed. NADPH-DQ reductase activity in brain Ms was 2.5-fold higher than NADPH-PQ reductase activity. The formation of PQ and DQ radicals was verified optically and observed directly by ESR spectroscopy in the NADPH-PQ and -DQ reductase reactions by brain Ms under anaerobic conditions. PQ- and DQ-induced superoxide formation was confirmed by the detection of DMPO-OOH ESR signals and followed by chemiluminescence (CL) of a Cypridina luciferin analogue (CLA). The kinetics and intensity of the CL were consistent with the observations that the reduction in DQ is faster than that in PQ. Thiobarbituric acid reactive substances (TBARS) and phospholipid hydroperoxides in brain Ms increased in the presence of NADPH and Fe3+. The generation of both lipid peroxidation products derived from brain Ms decreased with increasing concentrations of PQ and DQ. The inhibitory effect of DQ is more pronounced than that of PQ. The formation of PQ- and DQ-induced reactive oxygen species was not associated with lipid peroxidation in rat brain Ms.