Hyaluronic Acid Degradation by Ascorbic Acid and Influence of Iron

The effects of ascorbic acid, iron and ADP on hyaluronic acid, a compound present in inflamed joints, were investigated in an in vitro system. Ascorbic acid induces degradation of hyaluronic acid which increased in the presence of FeCl, and which is additionally stimulated by ADP chelated ferric ions. The hyaluronic acid degrading reactions induced by the Fe-III/ADP/ascorbic acid system were inhibited by catalase and formate to various extents whereas the presence of superoxide dismutase did not exert any inhibitory effect. Desferrioxamine, a specific iron chelator, completely inhibited hyaluronic acid depolymerisation by ascorbic acid as well as in combination with FeCl₃ or FeCl₃/ADP, respectively. We suggest that the ultimate hyaluronic acid degrading species is OH', generated via the Fe-III/ADP catalysed Haber Weiss reaction. There is also an indication for the involvement of perferryl or/and ferryl species in the degradation process.     

Iron prevents ascorbic acid (vitamin C) induced hydrogen peroxide accumulation in copper contaminated drinking water

Ascorbic acid (vitamin C) induced hydrogen peroxide (H₂O₂) formation was measured in household drinking water and metal supplemented Milli-Q water by using the FOX assay. Here we show that ascorbic acid readily induces H₂O₂ formation in Cu(II) supplemented Milli-Q water and poorly buffered household drinking water. In contrast to Cu(II), iron was not capable to support ascorbic acid induced H₂O₂ formation during acidic conditions (pH: 3.5-5). In 12 out of the 48 drinking water samples incubated with 2 mM ascorbic acid, the H₂O₂ concentration exceeded 400 μM. However, when trace amounts of Fe(III) (0.2 mg/l) was present during incubation, the ascorbic acid/Cu(II)-induced H₂O₂ accumulation was totally blocked. Of the other common divalent or trivalent metal ions tested, that are normally present in drinking water (calcium, magnesium, zinc, cobalt, manganese or aluminum), only calcium and magnesium displayed a modest inhibitory activity on the ascorbic acid/Cu(II)-induced H₂O₂ formation. Oxalic acid, one of the degradation products from ascorbic acid, was confirmed to actively participate in the iron induced degradation of H₂O₂. Ascorbic acid/Cu(II)-induced H₂O₂ formation during acidic conditions, as demonstrated here in poorly buffered drinking water, could be of importance in host defense against bacterial infections. In addition, our findings might explain the mechanism for the protective effect of iron against vitamin C induced cell toxicity.     

Iron (III) Stimulation of Lipid Hydroperoxide-Dependent Lipid Peroxidation

In an experimental system where both Fe²⁺ autoxidation and generation of reactive oxygen species is negligible, the effect of FeCl₂ and FeCl₃ on the peroxidation of phosphatidylcholine (PC) liposomes containing different amounts of lipid hydroperoxides (LOOH) was studied; Fe²⁺ oxidation, oxygen consumption and oxidation index of the liposomes were measured. No peroxidation was observed at variable FeCl₂/FeCl₃ ratio when PC liposomes deprived of LOOH by triphenyl-phosphine treatment were utilized. By contrast, LOOH containing liposomes were peroxidized by FeCl₂. The FeCl₂ concentration at which Fe²⁺ oxidation was maximal, defined as critical Fe²⁺ concentration [Fe²⁺]*, depended on the LOOH concentration and not on the amount of PC liposomes in the assay. The LOOH-dependent lipid peroxidation was stimulated by FeCl₃, addition; the oxidized form of the metal increased the average length of radical chains, shifted to higher values the [Fe²⁺]* and shortened the latent period. The iron chelator KSCN exerted effects opposite to those exerted by FeCl₃ addition. The experimental data obtained indicate that the kinetics of LOOH-dependent lipid peroxidation depends on the Fe²⁺/Fe³⁺ ratio at each moment during the time course of lipid peroxidation. The results confirm that exogenously added FeCl₃ does not affect the LOOH-independent but the LOOH-deendent lipid peroxidation; and suggest that the Feg, endogenously generated exerts a major role in the control of the LOOH-dependent lipid peroxidation.     

Iron (II) Ions Induced Oxidation of Ascorbic Acid and Glucose

Lipid peroxidation (LPO) of polyunsaturated fatty acids (PUFAs) is suspected to be involved in the generation of chronic diseases. A model reaction for LPO is the air oxidation of PUFAs initiated by Fe²⁺ and ascorbic acid. In the course of such model reactions glycolaldehyde (GLA) was detected as main aldehydic product. Since it is difficult to explain the generation of GLA by oxidation of PUFAs, it was suspected that GLA might be derived by oxidation of ascorbic acid. This assumption was verified by treatment of ascorbic acid with Fe²⁺.

Produced aldehydic compounds were trapped by addition of pentafluorobenzylhydroxylamine hydrochloride (PFBHA-HCl), trimethylsilylated and finally identified by gas chromatography/mass spectrometry (GC/MS). Oxidation of ascorbic acid with O₂ in presence of iron ions produced not only glycolaldehyde (GLA), but also glyceraldehyde (GA), dihydroxyacetone (DA) and formaldehyde. Glyoxal (GO) and malondialdehyde (MDA) were detected as trace compounds.

The yield of the aldehydic compounds was increased by addition of lipid hydroperoxides (LOOH) or H₂O₂. The buffer influenced the reaction considerably: Iron ions react with Tris buffer by producing dihydroxyace-tone (DA). Since ascorbic acid is present in biological systems and Fe²⁺ ions are obviously generated by cell damaging processes, the production of GLA and other aldehydic components might add to the damaging effects of LPO.

Glucose suffers also oxidation to short-chain aldehydic compounds in aqueous solution, but this reaction requires addition of equimolar amounts of Fe²⁺ together with equimolar amounts of H₂O₂ or 13-hydroperoxy-9-cis-11-trans-octadecadienoic acid (13-HPODE). Therefore this reaction, also influenced by the buffer system, seems to be not of biological relevance.     

The Generation of Ferryl or Hydroxyl Radicals During Interaction of Haemproteins with Hydrogen Peroxide

The oxidation of 2-keto-4-thiomethyl butyric acid (KTBA) and methionine to ethylene has been used to evaluate generation of ferryl species or hydroxyl radicals by H₂O₂-_-activated haemproteins or free ferric ions. Hydrogen peroxide was generated by a glucose oxidase-glucose system at a rate of 1 μM/min. Free ferric in the presence of H₂O₂ oxidizes KTBA, and this was highly inhibited by hydroxyl radical scavengers, caeruloplasmin, superoxide dismutase (SOD) and EDTA. However, when metmyoglobin, methaemoglobin (MtHb) or horseradish peroxidase (HRP) were tested in the same model system, hydroxyl radical scavengers suppressed partially KTBA oxidation and caeruloplasmin, SOD and EDTA failed to inhibit the reaction. Cytochrome-c was found to be a weak promoter of KTBA oxidation in the presence of H₂O₂. Methionine was oxidized to ethylene by an active system which generates hydroxyl radicals, but not by H₂O₂-_-activated metmyoglobin. Ferric ions chelated to membranes or ADP in the presence of H₂O₂ generated enzymatically, initiated membranal lipid peroxidation only in the presence of ascorbic acid, and this was inhibited by EDTA. In contrast, metmyoglobin and methaemoglobin activated by H₂O₂ generated by the same system, initiated membranal lipid peroxidation and this was not inhibited by EDTA. It is concluded that ferryl and not HO. is the main oxidant in systems containing myoglobin and haemoglobin activated by low concentrations of H₂O₂.     

Intracellular Ascorbic Acid Inhibits the Na+-Ca2+ Exchanger in Cultured Rat Astrocytes

Abstract: The effect of ascorbic acid on Ca²⁺ uptake in cultured rat astrocytes was examined in the presence of ouabain and monensin, which are considered to drive the Na⁺-Ca²⁺ exchanger in the reverse mode. Ascorbic acid at 0.1–1 m M inhibited Na⁺-dependent Ca²⁺ uptake significantly but not Na⁺-dependent glutamate uptake in the cells, although the inhibition required pretreatment for more than 30 min. The effect of ascorbic acid on the Ca²⁺ uptake was blocked by simultaneous addition of ascorbate oxidase (10 U/ml). Na⁺-dependent Ca²⁺ uptake was also inhibited by isoascorbate at 1 m M but not by ascorbate 2-sulfate, dehydroascorbate, and sulfhydryl-reducing reagents such as glutathione and 2-mercaptoethanol. The inhibitory effect of ascorbic acid was observed even in the presence of an inhibitor of lipid peroxidation, o -phenanthroline, or a radical scavenger, mannitol, and the degrading enzymes such as catalase and superoxide dismutase. On the other hand, the inhibitory effect was not observed under the Na⁺-free conditions that inhibited the uptake of ascorbic acid in astrocytes. When astrocytes were cultured for 2 weeks in a medium containing ascorbic acid, the content of ascorbic acid in the cells was increased and conversely Na⁺-dependent Ca²⁺ uptake was decreased. These results suggest that an increase in intracellular ascorbic acid results in a decrease of Na⁺-Ca²⁺ exchange activity in cultured astrocytes and the mechanism is not related to lipid peroxidation.     

Kinetics of the Competitive Degradation of Deoxyribose and Other Molecules by Hydroxyl Radicals Produced by the Fenton Reaction in the Presence of Ascorbic Acid

The competition method in which the Fenton reaction is employed as an OH radical generator and deoxyribose as a detecting molecule, has been used to determine the rate constants for reactions of the OH radical with its scavengers. Nonlinear competition plots were obtained for those scavengers which reacted with the Fenton reagents (Fe²⁺ or H₂O₂). Ascorbic acid is believed to overcome this problem. We have investigated the kinetics of deoxyribose degradation by -OH radicals generated by the Fenton reaction in the presence of ascorbic acid, and observed that the inclusion of ascorbic acid in the Fenton system greatly increased the rate of OH radical generation. As a result, the interaction between some scavengers and the Fenton reagents became negligeable and linear competition plots of A7A vs scavenger concentrations were obtained. The effects of experimental conditions such as, the concentrations of ascorbic acid, deoxyribose, H₂O₂ and Fe²⁺-EDTA, the EDTA/Fe²⁺ ratio as well as the incubation time, on the deoxyribose degradation and the determination of the rate constant for mercaptoethanol chosen as a reference compound were studied. The small standard error, (6.76± 0.21) ±' 10⁹M^-1s^-1 observed for the rate constant values for mercaptoethanol determined under 13 different experimental conditions, indicates the latter did not influence the rate constant determination. This is in fact assured by introducing a term, k_x, into the kinetic equation. This term represents the rate of-OH reactions with other reagents such as ascorbic acid, Fe²⁺-EDTA, H₂O₂ etc. The agreement of the rate constants obtained in this work with that determined by pulse radiolysis techniques for cysteine, thiourea and many other scavengers, suggests that this simple competition method is applicable to a wide range of compounds, including those which react with the Fenton reagents and those whose solubility in water is low.     

Lipid Peroxidation. Definition of Experimental Conditions for Selective Study of the Propagation and Termination Phases

To find experimental conditions to selectively study the propagation phase of lipoperoxidation we studied the lipoperoxidation, catalyzed by FeCl₂, of liposomes in a buffering condition where Fe²⁺ autoxidation and oxygen active species generation does not occur. Liposomes from egg yolk phosphatidylcholine. prepared by vortex mixing, do not oxidize Fe²⁺: on the contrary they oxidize Fe²⁺ when prepared by ultrasonic irradiation. Dimyristoyl phosphatidylcholine liposomes prepared by ultrasonic irradiation do not oxidize Fe²⁺. During sonication polyunsaturated fatty acid residues autoxidize and lipid hydroperoxides (LOOH) are generated. Only when LOOH are present in the liposimes Fe²⁺ oxidizes and its rate of oxidation depends on the amount of LOOH in the assay. The reaction results in the generation of both LOOH and thiobarbituric acid reactive material (TBAR): it is inhibited by butylated hydroxytoluene and has a acidic pH optimum; it is not inhibited by catalase and OH' scavengers. The reaction studied. thus, appears to be the chain branching and propagation phase of lipoperoxidation. When we studied the dependence of Fe²⁺ oxidation, LOOH and TBAR generation on FeCl₂ concentration, we observed that at high FeCl₂ concentrations the termination phase of lipoperoxidation was prevalent. Thus. by selecting the appropriate FeCl₂ concentration the proposed experimental system allows study of either the propagation or the termination phase of lipoperoxidation.     

Ascorbic acid as negative effector of the peroxidase-catalyzed degradation of indole-3-acetic acid

Ascorbic acid is a strong inhibitor of indole-3-acetic oxidation catalyzed by commercial horse-radish peroxidase. In the presence of excess ascorbic acid, the indole-acetic acid oxidation catalysis is apparently blocked. The activity of peroxidase for indoleacetic acid at pH 3.7 and 33°C, in the presence of 2,4-dichlorophenol and MnCl₂ as promotors was measured by polarographic technique. The K_m was 0.27 m M and the maximum velocity was 1.02 mmol O₂ (mg protein)⁻¹ min⁻¹. Dixon plots lead to an apparent K_i of 1.25 (μ M for ascorbic acid and the inhibition was apparently competitive. Ascorbic acid, besides appearing to be a strong inhibitor of the IAA oxidase activity of peroxidase, seemed to protect IAA from total degradation. Addition of more than 5 μ M ascorbic acid produced both an exponential increase in the lag time before the onset of reaction and, at the end, an oxidation protection of 26 μ M IAA when 111 μ M IAA was present at the stawrt. The possibility of ascorbic acid-IAA auxin from endogenous oxidation in plants, is proposed.     

The Action of Hydrogen Peroxide on the Formation of Thiobarbituric Acid-Reactive Material From Microsomes, Liposomes Or From Dna Damaged By Bleomycin Or Phenanthroline. Artefacts in the Thiobarbituric Acid Test

Incubation of rat-liver microsomes, previously azide-treated to inhibit catalase, with H₂O₂ caused a loss of cytochrome P-450 but not of cytochrome b₅. This loss of P-450 was not prevented by scavengers of hydroxyl radical, chain-breaking antioxidants or metal ion-chelating agents. Application of the thiobarbituric acid (TBA) assay to the reaction mixture suggested that H₂O₂ induces lipid peroxidation, but this was found to be due largely or completely to an effect of H²O₂ on the TBA assay. By contrast, addition of ascorbic acid and Fe(III) to the microsomes led to lipid peroxidation and P-450 degradation: both processes were inhibited by chelating agents and chain-breaking antioxidants, but not by hydroxyl radical scavengers. H₂O₂ inhibited ascorbate/Fe (III)-induced microsomal lipid peroxidation, but part of this effect was due to an action of H₂O₂ in the TBA test itself. H₂O₂ also decreased the colour measured after carrying out the TBA test upon authentic malondialdehyde, tetraethoxypropane, a DNA-Cu²⁺/o-phenanthroline system in the presence of a reducing agent, ox-brain phospholipid liposomes in the presence of Fe(III) and ascorbate, or a bleomycin-iron ion/DNA/ascorbate system. Caution must be used in interpreting the results of TBA tests upon systems containing H₂O₂.     

Plant Resistance Mechanisms to Air Pollutants: Rhythms in Ascorbic Acid Production During Growth Under Ozone Stress

Relationships between ozone (O₃) tolerance and leaf ascorbic acid concentrations in 0₃-susceptible (O₃S) 'Hark' and O₃-resistant (O₃R) 'Hood'soybean, Glycine max (L.) Merr., cultivars were examined with high-performance liquid chromatography (HPLC). Leaf samples were analyzed at 4 h intervals during a 24 h period. Soybean cultivars grown in the greenhouse with charcoal filtered (CF) and nonfiltered (NF) air showed daily oscillations in ascorbic acid production. Highest ascorbic acid levels in leaves during light coincided with highest concentrations of photochemical oxidants in the atmosphere at 2:00 p.m. The resistant genotype produced more ascorbic acid in its trifoliate leaves than did the corresponding susceptible genotype. Under CF air (an O₃-reduced environment) O₃-S and O₃-R cultivars showed rhythms in ascorbic acid production. In NF air (an O₃ stress environment) the O₃-R cultivar alone showed rhythms in ascorbic acid production. Results indicated that superior O₃ tolerance in the Hood soybean cultivar (compared with Hark) was associated with a greater increase in endogenous levels of ascorbic acid. Ascorbic acid may scavenge free radicals and thereby protect cells from injury by O₃ or other oxyradical products. Plants defend themselves against photochemical oxidant stress, such as O₃, by several mechanisms. Experimental evidence indicates that antioxidant defense systems existing in plant tissues may function to protect cellular components from deleterious effects of photochemical oxidants through endogenous and exogenous controls.     

Haemoglobin and Myoglobin as Inhibitors of Hydroxyl Radical Generation in a Model System of “Iron Redox” Cycle

Methionine was oxidized to ethylene by an “Iron Redox” system containing H₂O₂, Fe-EDTA and ascorbate. generating hydroxyl radicals or another species of similar reactivity. Oxy or met forms of haemoglobin and myoglobin were found to inhibit methionine oxidation. Methionine oxidation was elevated in the “Iron Redox” system by increasing ascorbic acid concentration. However, in the presence of metmyoglobin or methaemoglobin, the increases in ascorbic acid did not lower the haemproteins' inhibitory effects but rather increased them.

The pro-oxidative or anti-oxidative activities of haemproteins in biological oxidative reactions seem to be dependent on compartmentalization and on the presence and concentrations of reducing compounds and H₂O₂.     

Degradation of Hyaluronic Acid by Photosensitized Riboflavin In Vitro. Modulation of the Effect by Transition Metals, Radical Quenchers, and Metal Chelators

The effect of photoexcited riboflavin (RF) on the viscosity of hyaluronic acid (HA) solutions has been investigated. UV irradiation of RF causes under aerobic conditions fragmentation of HA and a decrease in the viscosity of its solutions. A decrease of HA viscosity occurs in PO₄-buffered solutions and is accelerated by high pH, Fe²⁺ (but much less so by Fe³⁺), certain metal chelators, and horseradish peroxidase (HRP); it is partially inhibited by catalase and less so by superoxide dismutase (SOD). The reactivity of the system was completely blocked by Tris, ethanol, aspirin, d-manitol, dimethylthiourea (DMTU), dimethylsulfoxide (DMSO), and sodium azide. These results indicate that the most likely chemical species involved in the reaction is the hydroxyl radical. Singlet oxygen (¹0₂) generation is suggested by the ability of NaN₃ and DMSO to completely inhibit the reactivity of the system. These two agents, however, may also interact with OH^√ radical, as well and suppress the reactivity of the system. H₂O₂ and seem also to be produced in significant amounts, because catalase and SOD partially block the reactivity of the system. The effect of HRP may be due to hydrogen subtraction from HA and H₂O₂ reduction to water. Photoexcitation of RF may potentially occur in vitro and in vivo in the organs and tissues that are permeable to light, such as the eye or skin, and damage HA and other cell-matrix components causing inflammation and accelerating aging.  © 1997 Elsevier Science Inc.     

Cysteine Sulfinic Acid in the Central Nervous System: Uptake and Release of Cysteine Sulfinic Acid by a Rat Brain Preparation

Abstract: Uptake and release of cysteine sulfinic acid by synaptosomal fractions (P₂) and slices of rat cerebral cortex were investigated. The P₂ fraction had a Na⁺-dependent high-affinity uptake system for cysteine sulfinic acid (K_m, 12μM), which was restricted to the synaptosomes. High-affinity uptake of cysteine sulfinic acid was competitively inhibited by glutamate, aspartate, and cysteic acid. None of the various centrally acting drugs tested specifically inhibited this transport system. Release of [¹⁴C]cysteine sulfinic acid from preloaded cortical slices or P₂ fractions was examined by a superfusion method, which avoided reuptake of released [¹⁴C]cysteine sulfinic acid. High K⁺ (56 m M ) and veratridine (10μM) stimulated the release of cysteine sulfinic acid from slices and the P₂ fraction in a partly Ca²⁺-dependent manner. Diazepam at concentrations of 10 and 100 μM markedly inhibited the stimulated release, but not the spontaneous release, by cortical slices. On the contrary, it had no effect on the stimulated release of cysteine sulfinic acid from the P₂ fraction.     

Ascorbic acid catabolism by gut microflora: studies in germ-free and conventional guinea pigs

The role of gut microflora in ascorbic acid catabolism was investigated in both conventional and germ-free guinea pigs. In vitro studies demonstrated extensive degradation of the vitamin by fresh feces, cecal, and colonic contents of conventional guinea pigs. Direct injection of [1-¹⁴C] ascorbic acid into the cecum of conventional guinea pigs in vivo yielded a 70% recovery of the label as respiratory ¹⁴CO₂ within 6 hr compared with only 5% recovery following injection into the virtually sterile peritoneum in a comparable group of conventional guinea pigs. Thus, ascorbic acid not absorbed prior to reaching the lower gastrointestinal tract stands to be extensively decarboxylated by microflora in the cecum. In a companion study of germ-free guinea pigs, 10% of an administered dose of [1-¹⁴C] ascorbic acid was expired as ¹⁴CO₂ within 36 hr post-injection following intraperitoneal injection compared with 16% recovery in a matched group of conventional animals injected at the same site. Results of this series of studies suggest that hepatic decarboxylation and gut microflora, in tandem, contribute to ascorbic acid decarboxylation in this species.     

Melatonin reduces lipid and protein oxidative damage in synaptosomes due to aluminium

Prolonged exposure to excessive aluminium (Al) concentrations is involved in the ethiopathology of certain dementias and neurological disorders. Melatonin is a well-known antioxidant that efficiently reduces lipid peroxidation due to oxidative stress. Herein, we investigated in synaptosomal membranes the effect of melatonin in preventing Al promotion of lipid and protein oxidation when the metal was combined with FeCl₃ and ascorbic acid. Lipid peroxidation was estimated by quantifying malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA) concentrations in the membrane suspension and protein carbonyls were measured in the synaptosomes as an index of oxidative damage. Under our experimental conditions, the addition of Al (0.0001–1 mmol/L) enhanced MDA+4-HDA formation in the synaptosomes. In addition, Al (1 mmol/L) raised protein carbonyl contents. Melatonin reduced, in a concentration-dependent manner, lipid and protein oxidation due to Al, FeCl₃ and ascorbic acid in the synaptosomal membranes. These results show that melatonin confers protection against Al-induced oxidative damage in synaptosomes and suggest that this indoleamine may be considered as a neuroprotective agent in Al toxicity because of its antioxidant activity.     

In vitro nonenzymatic glycation enhances the role of myoglobin as a source of oxidative stress

Metmyoglobin (Mb) was glycated by glucose in a nonenzymatic in vitro reaction. Amount of iron release from the heme pocket of myoglobin was found to be directly related with the extent of glycation. After in vitro glycation, the unchanged Mb and glycated myoglobin (GMb) were separated by ion exchange (BioRex 70) chromatography, which eliminated free iron from the protein fractions. Separated fractions of Mb and GMb were converted to their oxy forms -MbO₂ and GMbO₂, respectively. H₂O₂-induced iron release was significantly higher from GMbO₂ than that from MbO₂. This free iron, acting as a Fenton reagent, might produce free radicals and degrade different cell constituents. To verify this possibility, degradation of different cell constituents catalyzed by these fractions in the presence of H₂O₂ was studied. GMbO₂ degraded arachidonic acid, deoxyribose and plasmid DNA more efficiently than MbO₂. Arachidonic acid peroxidation and deoxyribose degradation were significantly inhibited by desferrioxamine (DFO), mannitol and catalase. However, besides free iron-mediated free radical reactions, role of iron of higher oxidation states, formed during interaction of H₂O₂ with myoglobin might also be involved in oxidative degradation processes. Formation of carbonyl content, an index of oxidative stress, was higher by GMbO₂. Compared to MbO₂, GMbO₂ was rapidly auto-oxidized and co-oxidized with nitroblue tetrazolium, indicating increased rate of Mb and superoxide radical formation in GMbO₂. GMb exhibited more peroxidase activity than Mb, which was positively correlated with ferrylmyoglobin formation in the presence of H₂O₂. These findings correlate glycation-induced modification of myoglobin and a mechanism of increased formation of free radicals. Although myoglobin glycation is not significant within muscle cells, free myoglobin in circulation, if becomes glycated, may pose a serious threat by eliciting oxidative stress, particularly in diabetic patients.     

Inhibitory Effects of Ascorbic Acid on the Binding of [3H]Dopamine Antagonists to Neostriatal Membrane Preparations : Relationship to Lipid Peroxidation

Abstract: Ascorbic acid, sodium ascorbate, and isoascorbic acid (the stereo-isomer of ascorbic acid) inhibited the stereospecific binding of [³H]spiroperidol to neostriatal membrane preparations. Greater inhibitory effects were obtained at intermediate concentrations of the three ascorbic acid analogs (i.e., 0.06 and 0.6 mM) than at higher (6 m M ) or lower (0.006 m M ) concentrations. In parallel experiments, the three ascorbic acid analogs induced lipid peroxidation, which was also greater at the two intermediate than at higher or lower concentrations. Several known inhibitors of lipid peroxidation, including propyl gallate, butylated hydroxyanisole, butylated hydroxytoluene, α-naphthol, and cobalt chloride, as well as the iron chelating agents EDTA and DETAPAC (diethylenetriaminepentaacetic acid) were able to counteract the effects of the ascorbic acid analogs on both lipid peroxidation and on [³H]spiroperidol binding. These data strongly suggest that an iron-catalyzed lipid peroxidation is responsible for the observed inhibitory effects on binding. In other experiments, neostriatal membrane preparations that were preincubated with ascorbic acid (0.6 m M ) and subsequently washed still had greatly diminished capacity to bind [³H]spiroperidol, indicating that ascorbic acid need not be physically present during the binding assay in order to affect binding. This experimental procedure also appears to be a way in which [³H]spiroperidol binding sites can be inactivated and washed free of the inactivating agent.     

Ascorbic Acid Inhibits Lipid Peroxidation but Enhances DNA Damage in Rat Liver Nuclei Incubated with Iron Ions

In this report we studied DNA damage and lipid peroxidation in rat liver nuclei incubated with iron ions for up to 2 hrs in order to examine whether nuclear DNA damage was dependent on membrane lipid peroxidation. Lipid peroxidation was measured as thio-barbituric acid-reactive substances (TBARS) and DNA damage was measured as 8-OH-deoxyguanosine (8-OH-dG). We showed that Fe(II) induced nuclear lipid peroxidation dose-dependently but only the highest concentration (1.0 mM) used induced appreciable 8-OH-dG. Fe(II1) up to 1 mM induced minimal lipid peroxidation and negligible amounts of 8-OH-dG. Ascorbic acid enhanced Fe(II)-induced lipid peroxidation at a ratio to Fe(II) of 1:l but strongly inhibited peroxidation at ratios of 2.5:l and 5:l. By contrast, ascorbate markedly enhanced DNA damage at all ratios tested and in a concentration-dependent manner. The nuclear DNA damage induced by 1 niM FeSO₄/5 mM ascorbic acid was largely inhibited by iron chelators and by dimethylsulphoxide and manni-tol, indicating the involvement of OH. Hydrogen peroxide and superoxide anions were also involved, as DNA damage was partially inhibited by catalase and, to a lesser extent, by superoxide dismutase. The chain-breaking antioxidants butylated hydroxytoluene and diphenylamine (an alkoxyl radical scavenger) did not inhibit DNA damage. Hence, this study demonstrated that ascorbic acid enhanced Fe(II)-induced DNA base modification which was not dependent on lipid peroxidation in rat liver nuclei.