ascorbic-acid and ferric-nitrilotriacetate

Ascorbic acid (AA) is a naturally occurring phenolic compound with antioxidant properties used in food, cosmetics, and pharmaceutical products. In this study, the effect of AA on ferric nitrilotriacetate (Fe-NTA)-induced hepatotoxicity in rats has been examined. Fe-NTA alone enhances ornithine decarboxylase activity to 4.5-fold and tritiated thymidine incorporation in DNA to 3.6-fold in livers compared with the corresponding saline-treated controls. The enhanced ornithine decarboxylase activity and DNA synthesis showed a reduction to 3.02- and 1.88-fold, respectively, at a higher dose of 2 mg AA per day per animal, compared with the Fe-NTA-treated groups. Fe-NTA treatment also enhanced the hepatic microsomal lipid peroxidation to 1.7-fold compared to saline-treated controls. These changes were reversed significantly in animals receiving pretreatment of AA. The present data shows that AA can reciprocate the toxic effects of Fe-NTA and can serve as a potent chemopreventive agent to suppress oxidant-induced tissue injury and hepatotoxicity in rats.

Topics: Animals; Antioxidants; Ascorbic Acid; Chemical and Drug Induced Liver Injury; DNA; Ferric Compounds; L-Lactate Dehydrogenase; Lipid Peroxidation; Liver; Male; Nitrilotriacetic Acid; Ornithine Decarboxylase; Oxidative Stress; Protein Carbonylation; Rats; Transaminases

Ascorbic acid (AA) is a naturally occurring organic compound with antioxidant properties. It is necessary for normal growth and development, and has been shown to protect against tissue toxicity and oxidative stress.. The protective effect of AA against nephrotoxicity induced in albino rats by ferric nitrilotriacetate (Fe-NTA) was evaluated.. Male albino rats of Wistar strain (4-6 weeks old) weighing 125-150 g were used in this study. Animals were given a single dose of Fe-NTA (9 mg/kg body weight, intraperitoneal) after a week of treatment with AA (1 and 2 mg/animal/day).. Fe-NTA treatment enhanced microsomal lipid peroxidation (LPO) and hydrogen peroxide (H2O2) generation to 1.7- to 2.2-fold, glutathione (GSH) levels were decreased by two-fold and the activities of GSH metabolizing enzymes decreased to a range of 2.2- to 2.5-fold in renal tissue. These changes were reversed significantly in animals receiving pretreatment of AA. Treatment of rats with AA prior to the treatment with Fe-NTA decreased microsomal LPO and H2O2 generation to 124 and 172%, and also resulted in the recovery of reduced levels of GSH, GSH-metabolizing enzymes to almost 92% at the higher dose level of AA.. AA protects against Fe-NTA-induced nephrotoxicity and renal damage. AA has a beneficial impact on Fe-NTA-induced toxicity due to its scavenging and antioxidant effect in albino rats.

Topics: Animals; Antioxidants; Ascorbic Acid; Ferric Compounds; Glutathione; Glutathione Peroxidase; Glutathione Transferase; Kidney Diseases; Lipid Peroxidation; Male; Microsomes; Nitrilotriacetic Acid; Oxidative Stress; Rats; Rats, Wistar

Most pomegranate (Punica granatum Linn., Punicaceae) fruit parts are known to possess enormous antioxidant activity. The present study evaluated antioxidant and hepatoprotective activity of pomegranate flowers. Alcoholic (ethanolic) extract of flowers was prepared and used in the present study. The extract was found to contain a large amount of polyphenols and exhibit enormous reducing ability, both indicative of potent antioxidant ability. The extract showed 81.6% antioxidant activity in DPPH model system. The ability of extract to scavenge reactive oxygen species (ROS) and reactive nitrogen species (RNS) was tested and it was found to significantly scavenge superoxide (O(2)(.-)) (by up to 53.3%), hydrogen peroxide (H(2)O(2)) (by up to 30%), hydroxyl radicals (()OH) (by up to 37%) and nitric oxide (NO) (by up to 74.5%). The extract also inhibited (.)OH induced oxidation of lipids and proteins in vitro. These results indicated pomegranate flower extract to exert a significant antioxidant activity in vitro. The efficacy of extract was tested in vivo and it was found to exhibit a potent protective activity in acute oxidative tissue injury animal model: ferric nitrilotriacetate (Fe-NTA) induced hepatotoxicity in mice. Intraperitoneal administration of 9 mg/kg body wt. Fe-NTA to mice induced oxidative stress and liver injury. Pretreatment with pomegranate flower extract at a dose regimen of 50-150 mg/kg body wt. for a week significantly and dose dependently protected against Fe-NTA induced oxidative stress as well as hepatic injury. The extract afforded up to 60% protection against hepatic lipid peroxidation and preserved glutathione (GSH) levels and activities of antioxidant enzymes viz., catalase (CAT), glutathione peroxidase (GPX) glutathione reductase (GR) and glutathione-S-transferase (GST) by up to 36%, 28.5%, 28.7%, 40.2% and 42.5% respectively. A protection against Fe-NTA induced liver injury was apparent as inhibition in the modulation of liver markers viz., aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), bilirubin and albumin in serum. The histopathological changes produced by Fe-NTA, such as ballooning degeneration, fatty changes, necrosis were also alleviated by the extract. These results indicate pomegranate flowers to possess potent antioxidant and hepatoprotective property, the former being probably responsible for the latter.

Topics: Animals; Antioxidants; Ascorbic Acid; Biphenyl Compounds; Catalase; Chemical and Drug Induced Liver Injury; Ferric Compounds; Flowers; Free Radical Scavengers; Glutathione; Glutathione Peroxidase; Glutathione Transferase; Hydrogen Peroxide; Hydroxyl Radical; Lipid Peroxidation; Liver; Liver Function Tests; Lythraceae; Male; Mice; Nitric Oxide; Nitrilotriacetic Acid; Oxidants; Oxidation-Reduction; Oxidoreductases; Phenols; Picrates; Plant Extracts; Subcellular Fractions; Superoxides

Previous work from our laboratory demonstrated that pyridoxal isonicotinoyl hydrazone (PIH) has in vitro antioxidant activity against iron plus ascorbate-induced 2-deoxyribose degradation due to its ability to chelate iron; the resulting Fe(III)-PIH(2) complex is supposedly unable to catalyze oxyradical formation. A putative step in the antioxidant action of PIH is the inhibition of Fe(III)-mediated ascorbate oxidation, which yields the Fenton reagent Fe(II) [Biochim. Biophys. Acta 1523 (2000) 154]. In this work, we demonstrate that PIH inhibits Fe(III)-EDTA-mediated ascorbate oxidation (measured at 265 nm) and the formation of ascorbyl radical (in electron paramagnetic resonance (EPR) studies). The efficiency of PIH against ascorbate oxidation, ascorbyl radical formation and 2-deoxyribose degradation was dose dependent and directly proportional to the period of preincubation of PIH with Fe(III)-EDTA. The efficiency of PIH in inhibiting ascorbate oxidation and ascorbyl radical formation was also inversely proportional to the Fe(III)-EDTA concentration in the media. When EDTA was replaced by the weaker iron ligand nitrilotriacetic acid (NTA), PIH was much more effective in preventing ascorbate oxidation, ascorbyl radical formation and 2-deoxyribose degradation. Moreover, the replacement of EDTA with citrate, a physiological chelator with a low affinity for iron, also resulted in PIH having a higher efficiency in inhibiting iron-mediated ascorbate oxidation and 2-deoxyribose degradation. These results demonstrate that PIH removes iron from EDTA (or from either NTA or citrate), forming an iron-PIH complex that cannot induce ascorbate oxidation effectively, thus inhibiting iron-mediated oxyradical formation. These results are of pharmacological relevance because PIH has been considered for experimental chelating therapy in iron-overload diseases.

Topics: Ascorbic Acid; Chelating Agents; Deoxyribose; Edetic Acid; Electron Spin Resonance Spectroscopy; Ferric Compounds; Free Radicals; Hydroxyl Radical; Isoniazid; Nitrilotriacetic Acid; Oxidation-Reduction; Oxidative Stress; Pyridoxal

The Na(+)/H(+) exchanger is the main intracellular pH (pH(i)) regulator in hepatic stellate cells (HSCs) and plays a key role in regulating proliferation and gene expression. We evaluated the effect of specific inhibition of this exchanger on HSC proliferation and collagen synthesis in vivo and in vitro.. Rat HSCs were incubated in the presence of platelet-derived growth factor (PDGF), transforming growth factor (TGF)-beta1, iron ascorbate (FeAsc), and ferric nitrilotriacetate solution (FeNTA) with or without the Na(+)/H(+) exchanger inhibitor 5-N-ethyl-N-isopropyl-amiloride (EIPA). pH(i) and Na(+)/H(+) exchanger activity, cell proliferation, and type I collagen accumulation were measured by using the fluorescent dye 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein, by immunohistochemistry for bromodeoxyuridine, and by enzyme-linked immunosorbent assay, respectively. In vivo liver fibrosis was induced by dimethylnitrosamine administration and bile duct ligation (BDL) in rats treated or not treated with amiloride.. PDGF, FeAsc, and FeNTA increased Na(+)/H(+) exchange activity and induced HSC proliferation. TGF-beta1 had no effect on the Na(+)/H(+) exchanger and was able, as for FeAsc and FeNTA, to induce type I collagen accumulation. EIPA inhibited all the effects determined by PDGF, FeAsc, and FeNTA and had no effect on TGF-beta1-induced collagen accumulation. In vivo, amiloride reduced HSC proliferation, activation, collagen deposition, and collagen synthesis.. The Na(+)/H(+) exchanger can play a key role in the development of liver fibrosis and in HSC activation in vivo.

Topics: Amiloride; Animals; Anti-Arrhythmia Agents; Antineoplastic Agents; Ascorbic Acid; Carcinogens; Cell Division; Collagen; Diuretics; Ferric Compounds; Ferrous Compounds; Gene Expression; Hydrogen-Ion Concentration; In Situ Nick-End Labeling; Liver; Liver Cirrhosis; Male; Nitrilotriacetic Acid; Procollagen; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; RNA, Messenger; Sodium-Hydrogen Exchangers; Thymidine Phosphorylase; Transforming Growth Factor beta

This study examined some of the variables determining the efficiency of lipid peroxidation in egg yolk phosphatidylcholine liposomes and in microsomes exposed to enzymatically-generated superoxide radicals. The initiation of peroxidation required the presence of preformed lipid peroxides and a chelated metal catalyst. Comparison of the relative effectiveness of four iron chelating agents showed that the chelate must bind to the membrane by coulombic attraction between the charged membrane and a chelate carrying an opposite net charge. Of the chelates tested, only the carcinogenic ferric nitrilotriacetate [corrected] (Fe(3+)-NTA) was an effective catalyst of oxidation of all membranes, whether carrying a net charge, or not. We postulate that the unique catalytic capacity of the ferric nitrilotriacetate [corrected] (Fe(3+)-NTA) can be explained by its existence in two forms at neutral pH, each binding to oppositely charged membranes and initiating their peroxidation. This gives the complex the unique ability to bind to any membrane, which may be a factor in its carcinogenicity.

Topics: Animals; Ascorbic Acid; Carcinogens; Ferric Compounds; Hydrogen-Ion Concentration; Iron Chelating Agents; Lipid Peroxidation; Liposomes; Male; Microsomes, Liver; NADP; Nitrilotriacetic Acid; Oxidation-Reduction; Oxygen; Phosphatidylcholines; Rats; Rats, Wistar; Superoxides; Thiobarbituric Acid Reactive Substances

Tissue slices are a useful biological system for lipid peroxidation studies but their use for DNA damage studies is not well characterized. Hence, the present study investigates DNA damage in rat liver slices, in comparison with isolated rat liver nuclei and HepG2 human hepatoma cells, incubated with ferric nitrilotriacetate (Fe(III)-NTA), bromotrichloromethane (BrCCl(3)), bromobenzene (BrB) or 2-nitropropane (2-NP) at 37 degrees C for 2 hr. DNA damage was measured in slices, cells or nuclei after centrifugation as formation of as 8-hydroxy-2'-deoxyguanosine (8-OH-dGu) and loss of double-stranded (dsDNA) due to strand breakage using a fluorometric analysis of DNA unwinding (FADU). Lipid peroxidation was measured as thiobarbituric acid-reactive substances (TBARS) released into the medium. The results show that in liver slices and isolated nuclei, Fe/NTA (1 mM/4 mM) induced high levels of TBARS but low levels of 8-OH-dGu, whereas the oxidant induced low levels of TBARS and no formation of 8-OH-dGu in HepG2 cells. In all three systems, inclusion of ascorbate caused dose-dependent formation of 8-OH-dGu, and the levels were similar between liver slices and HepG2 cells but were far higher in isolated nuclei. In liver slices the FADU assay was not applicable due to limited solubilization of DNA from the slice, whereas the assay detected significant loss of dsDNA in HepG2 cells and slight loss in isolated nuclei induced by Fe/NTA with or without ascorbate. Liver slices incubated with 1 mm BrCCl(3), BrB or 2-NP had elevated TBARS but had little or no formation of 8-OH-dGu; none of these oxidants induced lipid peroxidation or DNA damage in HepG2 cells. When liver slices obtained from rats injected with diethylmaleate (to deplete GSH) were incubated with BrCCl(3), BrB or 2-NP, levels of TBARS and 8-OH-dGu increased markedly. Similarly, HepG2 cells with decreased GSH showed marked elevation of TBARS and loss of dsDNA induced by these oxidants, although no formation of 8-OH-dGu was detected. The present study demonstrates the usefulness and limitations of liver slices for DNA damage studies and the importance of cellular GSH in the protection of DNA against environmental toxicants.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Ascorbic Acid; Bromobenzenes; Bromotrichloromethane; Carcinogens; Cell Nucleus; Deoxyguanosine; DNA Damage; Ferric Compounds; Humans; Lipid Peroxidation; Liver; Liver Neoplasms, Experimental; Nitrilotriacetic Acid; Nitroparaffins; Oxidation-Reduction; Propane; Rats; Thiobarbituric Acid Reactive Substances; Tumor Cells, Cultured

A high incidence of cancer has been correlated with chronic iron overload, and carotenoids are of interest as possible anticarcinogens. We have investigated the effect of lycopene on lipid peroxidation and on the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in CV1-P monkey cells exposed to ferric nitrilotriacetate (Fe-NTA) plus ascorbate. Cells supplemented with lycopene (20 pmol/10(6) cells) showed a reduction of 86% in Fe-NTA/ascorbate-induced lipid peroxidation (TBARS). Levels of 8-oxodGuo rose from 1.59+/-0.09 residues/10(6) dGuo in the control cells to 14.02+/-0.41 residues/10(6) dGuo after incubation with (1:4 mM) Fe-NTA/ascorbate (40 microM). Lycopene supplementation decreased in 77% the 8-oxodGuo levels in Fe-NTA/ascorbate-treated cells. These results indicate that lycopene can protect mammalian cells against membrane and DNA damage and possibly play a protective role against tumor promotion associated with oxidative damage.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Antioxidants; Ascorbic Acid; Carotenoids; Cell Line; Deoxyguanosine; DNA Damage; Ferric Compounds; Lipid Peroxidation; Lycopene; Mutagens; Nitrilotriacetic Acid; Oxidative Stress

The process of iron (Fe) release from cells plays an important role in health and disease, although the mechanisms responsible remain unclear. In this study we have examined the process of Fe efflux from HepG2 cells, including the possible roles of Cp and ascorbate in this process. Recently, it has been suggested that Cp plays no role in Fe release but can increase Fe uptake by Fe-deficient HepG2 cells (Mukhopadhyay et al. Science 1998;279:714-7). However, this latter study used a nonphysiologically relevant Fe complex (iron 59-NTA) to label cells with 59Fe at a nonphysiologic temperature (25 degrees C) and Cp concentration (<100 microg/mL). Because of these problems, the experiments have been repeated by maintaining physiologic conditions and labeling cells with the physiologic Fe donor diferric Tf. When cells were labeled at 37 degrees C with 59Fe-Tf in the presence of a physiologically relevant Cp concentration (300 microg/mL), this latter protein had no effect on the uptake of 59Fe in control cells or in cells depleted of Fe by using desferrioxamine. In addition, when Fe-replete or Fe-depleted cells were incubated with 59Fe-NTA at 25 degrees C or 37 degrees C, Cp had no effect on 59Fe uptake compared with the control. When the effect of Cp (10-500 microg/mL) on 59Fe release was examined in cells prelabeled with 59Fe-Tf, a concentration-dependent increase in 59Fe efflux was observed, whereas BSA had no effect. However, in contrast to membrane-permeable Fe chelators that caused a marked increase in Fe release, the effect of Cp on Fe efflux was less impressive. To further investigate the mechanism of 59Fe mobilization, we compared 59Fe efflux among HepG2 cells, SK-Mel-28 melanoma cells, and SK-N-MC neuroblastoma cells. These studies demonstrated that 59Fe release was dependent on the incubation time with 59Fe-Tf, the cell line, and the reincubation temperature. Although 59Fe mobilization from cells was markedly temperature dependent, a range of metabolic inhibitors did not affect 59Fe release. Additional experiments showed that physiologic concentrations of ascorbate reduced 59Fe efflux, whereas glutathione had no effect. This study provides further evidence that Cp is involved in Fe mobilization but does not appear to affect Fe uptake from Tf or NTA.

Topics: Antimetabolites; Apoproteins; Ascorbic Acid; Biological Transport, Active; Cell Line; Ceruloplasmin; Ferric Compounds; Humans; Iron; Iron Radioisotopes; Kinetics; Nitrilotriacetic Acid; Temperature; Transferrin

Ferric nitrilotriacetate (Fe-NTA) is a potent nephrotoxic agent. In this communication, we show the modulatory effect of DL-alpha-tocopherol (Vitamin-E) on ferric nitrilotriacetate (Fe-NTA)-induced renal oxidative stress, toxicity and hyperproliferative response in rats. Fe-NTA-treatment enhances the susceptibility of renal microsomal membrane for iron-ascorbate-induced lipid peroxidation and hydrogen peroxide generation which are accompanied by a decrease in the activities of renal antioxidant enzymes, catalase, glutathione peroxidase, glutathione reductase and glutathione-S-transferase and depletion in the level of renal glutathione. Parallel to these changes, a sharp increase in blood urea nitrogen and serum creatinine has been observed. In addition, Fe-NTA-treatment also enhances renal ornithine decarboxylase activity (ODC) and increases [3H]thymidine incorporation in renal DNA. Prophylactic treatment of animals with Vit.E daily for 1 week prior to the administration of Fe-NTA resulted in the diminution of Fe-NTA-mediated damage. Enhanced susceptibility of renal microsomal membrane for lipid peroxidation induced by iron-ascorbate and hydrogen peroxide generation were significantly reduced (P < 0.05). In addition, the depleted level of glutathione and inhibited activities of antioxidant enzymes recovered to significant levels (P < 0.05). Similarly, the enhanced blood urea nitrogen and serum creatinine levels which are indicative of renal injury showed a reduction of about 50% at a higher dose of Vit.E. The pretreatment of rats with Vit.E reduced the Fe-NTA-mediated induction in ODC activity and enhancement in [3H]thymidine incorporation in DNA. The protective effect of Vit.E was dose dependent. In summary, our data suggest that Vit.E is an effective chemopreventive agent in kidney and may suppress Fe-NTA-induced renal toxicity.

Topics: Animals; Ascorbic Acid; Blood Urea Nitrogen; Carcinogens; Catalase; Creatinine; Ferric Compounds; Glutathione; Glutathione Peroxidase; Glutathione Reductase; Glutathione Transferase; Hydrogen Peroxide; Kidney; Kidney Diseases; Lipid Peroxidation; Microsomes; Nitrilotriacetic Acid; Ornithine Decarboxylase; Oxidative Stress; Rats; Vitamin E

Iron uptake from a low-molecular-weight chelate Fe(III)-nitriloacetate (Fe-NTA) by anaerobic protozoan parasite Tritrichomonas foetus was investigated and compared with that from iron-saturated lactoferrin and transferrin. The results showed that the iron uptake from Fe-NTA was saturable (Km = 2.7 microM, Vmax = 21.7 fmol. microg-1.min-1) and time, and temperature dependent, thus suggesting involvement of a membrane transport carrier. The accumulation of iron from 59Fe-NTA was inhibited by NaF and iron chelators. Amilorid and inhibitors of endosome acidification did not influence the process. Ascorbate stimulated the uptake while a membrane impermeable chelator of bivalent iron (bathophenanthroline disulfonic acid) was inhibitory, suggesting that prior to transport iron is reduced extracellularly. In accord with this assumption, the reduction of ferric to ferrous iron in the presence of intact T. foetus cells was demonstrated. Dynamics and properties of uptake of iron released from transferrin were similar to those from Fe-NTA, indicating involvement of common mechanisms. Iron uptake from lactoferrin displayed profoundly different characteristics consistent with receptor-mediated endocytosis. Metronidazole-resistant derivative of the investigated T. foetus strain showed marked deficiency in iron acquisition from Fe-NTA and transferrin while its iron uptake from lactoferrin was higher than that of the parent strain. The results presented show that T. foetus possesses at least two independent mechanisms that mediate acquisition of iron.

Topics: Amiloride; Ammonium Chloride; Animals; Antiprotozoal Agents; Ascorbic Acid; Cattle; Chloroquine; Ferric Compounds; Iron; Iron Chelating Agents; Lactoferrin; Nitrilotriacetic Acid; Oxidation-Reduction; Sodium Fluoride; Transferrin; Tritrichomonas foetus

This study shows that trophoblast cells in culture are able to take up 59Fe from both Fe(III)nitrilotriacetate (NTA) and Fe-ascorbate. Fe in the presence of ascorbate is assumed to be Fe(III) in equilibrium with Fe(II). Kinetic parameters for non-transferrin iron uptake are determined from initial rate experiments, yielding Vmax=366 pmol/mg protein/5 min and Km=0.96 microM for Fe(III)NTA and Vmax=4043 pmol/mg protein/5 min and Km= 1.3 microM for Fe-ascorbate. Since trophoblast cells in culture reduce extracellular Fe(III)CN, and uptake of 59Fe from Fe-ascorbate is higher than that from Fe(III)NTA, it is suggested that reduction of Fe(III) precedes uptake. Uptake of 59Fe from both Fe-ascorbate and Fe(III)NTA is inhibited by Fe(II)chelator ferrozine and membrane-impermeable Fe(III)CN, further supporting this hypothesis. Studies with microvillous membrane vesicles (MMV) and basal membrane vesicles (BMV) reveal the presence of a NADH-dependent ferrireductase. Reduction of Fe(III)CN follows Michaelis-Menten kinetics, both with respect to [NADH] and [Fe]. NADPH is ineffective as electron donor. The rate of Fe(III)CN reduction by BMV is 2.5 times higher compared to MMV, while Km values for Fe(III)CN and NADH are not significantly different. These results reveal that a transmembrane NADH-dependent ferrireductase plays a role in uptake of non-transferrin iron. The possibility that this enzyme system is involved in iron transfer across the basal membrane is discussed.

Topics: Adult; Ascorbic Acid; Cells, Cultured; Chorionic Villi; Female; Ferric Compounds; Ferricyanides; Ferrous Compounds; Ferrozine; FMN Reductase; Humans; Intracellular Membranes; Iron Chelating Agents; Iron Radioisotopes; NADH, NADPH Oxidoreductases; Nitrilotriacetic Acid; Pregnancy; Transferrin; Trophoblasts

The mechanism of the antioxidant action of thiopalmitic acid (SH-Pal) was examined in an in vitro system measuring ferric (Fe(III)-nitrilotriacetate (NTA)- and Fe(III)-NTA/ascorbic acid (AsA)-induced lipid peroxidation of rat liver phospholipid liposomes and microsomes. The extent of lipid peroxidation was determined by measuring thiobarbituric acid reactive substances (TBARS). SH-Pal and glutathione (GSH) scarcely stimulated the Fe(III)-NTA-induced lipid peroxidation in contrast with the mode of action, being similar to those produced by reducing-agent antioxidants such as cysteine and AsA. SH-Pal reduced iron similar to the action produced by AsA and cysteine, but not that of GSH under the same conditions. Also, the reduction of iron by SH-Pal did not exhibit a pH dependency. Similarly, microsomal lipid peroxidation and oxygen consumption induced by Fe(III)-NTA/AsA were inhibited by the addition of SH-Pal in a time and dose dependent fashion, but GSH and cysteine exhibited a lower protective action. Time course studies on TBARS formation and oxygen consumption indicated the ability of SH-Pal to inhibit initiation and propagation reactions. Moreover, the microsomal lipid peroxidation induced by Cumene hydroperoxide (CumOOH) was progressively suppressed by the addition of increasing amounts of SH-Pal. These findings suggest that the antioxidant action of SH-Pal is partly due to complete reduction of iron at a faster rate and inhibition of oxygen consumption during the progress of the peroxidation. Further, SH-Pal has a protective action against free radical damage by hydroperoxy radical.

Topics: Animals; Antioxidants; Ascorbic Acid; Ferric Compounds; Glutathione; Hydrogen-Ion Concentration; In Vitro Techniques; Lipid Peroxidation; Liposomes; Male; Microsomes, Liver; Nitrilotriacetic Acid; Oxygen Consumption; Palmitic Acids; Phospholipids; Rats; Rats, Wistar; Thiobarbituric Acid Reactive Substances

In recent years two mechanisms have been proposed for the production of DNA strand breaks in cells undergoing oxidative stress: (i) DNA attack by OH radical, produced by Fenton reaction catalyzed by DNA-bound iron; and (ii) DNA attack by calcium-activated nucleases, due to the increase of cytosolic and nuclear calcium induced by oxidative stress. We set out to investigate the participation of the former mechanism by detecting and quantifying 3'-phosphoglycolate, a 3' DNA terminus known to be formed by OH radical attack to the deoxyribose moiety, followed by sugar ring rupture and DNA strand rupture. These structures were found in DNA of monkey kidney cells exposed to hydrogen peroxide, iron nitrilotriacetate or ascorbate, all species known to favor a cellular pro-oxidant status. The method employed to measure 3' phosphoglycolate was the 32P-postlabeling assay. Repair time course experiments showed that it takes 10 h for 3'-phosphoglycolate to be removed from DNA. It was found that the DNA of both control cells and cells exposed to hydrogen peroxide had a very poor capacity of supporting in vitro DNA synthesis, catalyzed by DNA polymerase I. If the DNA was previously incubated with exonuclease III, an enzyme able to expose 3'-OH primers by removal of 3'-phosphoglycolate and 3'-phosphate termini the in vitro synthesis was substantially increased. This result shows that either of these termini are present at the break and that 3'-hydroxyl termini are virtually absent. At least 25% of the strand breaks exhibited 3'-phosphoglycolate termini as determined by the 32P-postlabeling assay, but due to the characteristic of the method this percentage is likely to be higher. These results favor the hypothesis that an oxidative agent generated by Fenton reaction is responsible for DNA strand breakage in cells undergoing oxidative stress.

Topics: Animals; Ascorbic Acid; Cell Line; Chlorocebus aethiops; Deoxyribose; DNA; DNA Damage; DNA Polymerase I; DNA Repair; Exodeoxyribonucleases; Ferric Compounds; Fibroblasts; Glycolates; Hydrogen Peroxide; Hydroxyl Radical; Mutagens; Nitrilotriacetic Acid; Oxidative Stress

The present study characterizes the transport of nontransferrin (non-Tf) iron by K562 cells. Accumulation of radiolabel by cells incubated with 55Fe-nitrilotriacetate (NTA) is a saturable process that is time and temperature dependent (Ea approximately 20 kcal/mol). Initial rate analysis of iron influx yields values of Vmax = 855 fmol/min/10(6) cells and apparent Km = 0.54 microM. NHCL4 and chloroquine, agents that block cellular acquisition of iron from Tf, do not interfere with assimilation from FeNTA, demonstrating that uptake is truly independent of the Tf-mediated pathway. Furthermore, the inactivation of this transport mechanism by limited proteolytic digestion on ice indicates that specific cell surface proteins are involved. The extent of radiolabel incorporation into heme and ferritin is the same regardless of whether K562 cells acquire iron from 55FeNTA via the cell surface mechanism or from 55Fe-Tf via receptor-mediated endocytosis. Unlike other Tf-independent iron transport pathways that have been described, the K562 cell transport mechanism is not inhibited by divalent cations such as Ni2+, Co2+, or Mn2+. Uptake from 55FeNTA can be blocked by Cu2+ but at concentrations > 1500-fold molar excess. However, Cd2+ is a fairly specific inhibitor of 55Fe uptake by K562 cells (IC50 approximately 50 microM). Additionally, the K562 cell transport mechanism is not Ca2+ dependent and does not appear to be regulated by extracellular iron salts, in contrast to features noted for non-Tf iron uptake by fibroblasts (Sturrock, A., Alexander, J., Lamb, J., Craven, C. M., and Kaplan, J. (1991) J. Biol. Chem. 265, 3139-3145; Kaplan, J., Jordan, I., and Sturrock, A. (1991) J. Biol. Chem. 266, 2997-3004). These unique characteristics of the K562 cell uptake mechanism suggest that multiple transport systems function in Tf-independent iron assimilation.

Topics: Ascorbic Acid; Biological Transport; Calcium; Ferric Compounds; Humans; Iron; Kinetics; Leukemia, Erythroblastic, Acute; Nitrilotriacetic Acid; Transferrin; Tumor Cells, Cultured

A certain iron chelate, ferric nitrilotriacetate (Fe3+-NTA) is nephrotoxic and also carcinogenic to the kidney in mice and rats, a distinguishing feature not shared by other iron chelates tested so far. Iron-promoted lipid peroxidation is thought to be responsible for the initial events. We examined its ability to initiate lipid peroxidation in vitro in comparison with that of other ferric chelates. Chelation of Fe2+ by nitrilotriacetate (NTA) enhanced the autoxidation of Fe2+. In the presence of Fe2+-NTA, lipid peroxidation occurred as measured by the formation of conjugated diene in detergent-dispersed linoleate micelles, and by the formation of thiobarbituric acid-reactive substances in the liposomes of rat liver microsomal lipids. Addition of ascorbic acid to Fe3+-NTA solution promoted dose-dependent consumption of dissolved oxygen, which indicates temporary reduction of iron. On reduction, Fe3+-NTA initiated lipid peroxidation both in the linoleate micelles and in the liposomes. Fe3+-NTA also initiated NADPH-dependent lipid peroxidation in rat liver microsomes. Although other chelators used (deferoxamine, EDTA, diethylenetriaminepentaacetic acid, ADP) enhanced autoxidation, reduction by ascorbic acid, or in vitro lipid peroxidation of linoleate micelles or liposomal lipids, NTA was the sole chelator that enhanced all the reactions.

Topics: Adenosine Diphosphate; Animals; Ascorbic Acid; Deferoxamine; Edetic Acid; Ferric Compounds; Iron Chelating Agents; Linoleic Acid; Linoleic Acids; Lipid Peroxidation; Liposomes; Membrane Lipids; Micelles; Microsomes, Liver; NADP; Nitrilotriacetic Acid; Oxidation-Reduction; Oxygen; Pentetic Acid; Rats; Rats, Inbred Strains

Topics: Animals; Ascorbic Acid; Biological Transport; Cations, Divalent; Duodenum; Ferric Compounds; Ferrous Compounds; Hypoxia; Intestinal Absorption; Intestinal Mucosa; Male; Mice; Microvilli; Nitrilotriacetic Acid; Oxidation-Reduction