Ascorbate-Induced Lipid Peroxidation and Inhibition of [3H] Adenosine Binding to Rat Brain Synaptosomes

Abstract

Lipid peroxidation induced by ascorbate inhibited [³H] adenosine binding to rat brain synaptosomes, probably via changes in polyunsaturated fatty acid composition.     

H2O2-Induced Cell Death in Human Glioma Cells: Role of Lipid Peroxidation and PARP Activation

underlying mechanism of ROS-induced cell injury remains to be defined. This study was undertaken to examine the role of lipid peroxidation and poly (ADP-ribose) polymerase (PARP) activation in H₂O₂-induced cell death in A172 cells, a human glioma cell line. H₂O₂ induced a dose- and time-dependent cell death. The cell death was prevented by thiols (dithiothreitol and glutathione), iron chelators (deferoxamine and phenanthroline), H₂O₂ scavengers (catalase and pyruvate), and a hydroxyl radical scavenger (dimethylthiourea). Antioxidants N,N-diphenyl-p-phenylenediamine (DPPD) and Trolox had no effect on the H₂O₂-induced cell death. Lipid peroxidation did not increase in human glioma cells exposed to H₂O₂. The PARP inhibitor 3-aminobenzamide prevented the cell death induced by H₂O₂. The PARP activity was increased by H₂O₂ and the H₂O₂ effect was prevented by 3-aminobenzamide, dithiothreitol, and phenanthroline. The ATP depletion induced by H₂O₂ was prevented by catalase, dithiothreitol, phenanthroline, and 3-aminobenzamide, but not by DPPD. These results indicate that the H₂O₂-induced cell death is mediated by PARP activation but not by lipid peroxidation in human glioma cells.     

Lipid Peroxidation by the Manganese Peroxidase of Phanerochaete chrysosporium Is the Basis for Phenanthrene Oxidation by the Intact Fungus

The manganese peroxidase (MnP) of Phanerochaete chrysosporium supported Mn(II)-dependent, H₂O₂-independent lipid peroxidation, as shown by two findings: linolenic acid was peroxidized to give products that reacted with thiobarbituric acid, and linoleic acid was peroxidized to give hexanal. MnP also supported the slow oxidation of phenanthrene to 2,2′-diphenic acid in a reaction that required Mn(II), oxygen, and unsaturated lipids. Phenanthrene oxidation to diphenic acid by intact cultures of P. chrysosporium occurred to the same extent that oxidation in vitro did and was stimulated by Mn. These results support a role for MnP-mediated lipid peroxidation in phenanthrene oxidation by P. chrysosporium.     

Nickel-induced Inhibition of Wheat Root Growth is Related to H2O2 Production, but not to Lipid Peroxidation

Effects of exogenous nickel (Ni: 10 and 200 μM) on growth, mitotic activity, Ni accumulation, H₂O₂ content and lipid peroxidation as well as the activities of various antioxidative enzymes, such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione peroxidase (GSH-Px) were investigated in wheat roots. A considerable Ni accumulation in the roots occurred at both the concentrations. Although Ni at 10 μM did not have any significant effect on root growth, it strongly inhibited the root growth at 200 μM. Mitotic activity in the root tips was not significantly affected by exposure of the seedlings to 10 μM Ni; however, it was almost completely inhibited at 200 μM treatment. Ni stress did not result in any significant changes in CAT and APX activities as well as lipid peroxidation. However, H₂O₂ concentration increased up to 82% over the control in the roots of seedlings exposed to 200 μM Ni. There was a significant decline in both SOD (50%) and GSH-Px (20–30%) activities in the roots when the seedlings were treated with 200 μM Ni. The results indicated that a strong inhibition of wheat root growth caused by Ni stress was not due to enhanced lipid peroxidation, but might be related to the accumulation of H₂O₂ in root tissue.     

Ascorbate-Induced Lipid Peroxidation and Inhibition of [3H]Spiroperidol Binding in Neostriatal Membrane Preparations

Sodium ascorbate caused an increased lipid peroxidation and a large decrement in [3H]spiroperidol binding in a rat neostriatal membrane preparation (preparation C). Both effects were greater at intermediate (0.05 and 0.5 mM) than at higher or lower ascorbate concentrations. In contrast, in another neostriatal membrane preparation (preparation A), there was no loss of [3H]spiroperidol binding and only a small increase in lipid peroxidation caused by ascorbate. However, both the ascorbate-induced increase in lipid peroxidation and loss of [3H]spiroperidol binding were greatly enhanced in preparation A by the addition of iron salts. In experiments designed to explore reasons for these apparent discrepancies, we discovered that the method of tissue preparation was a critical factor. The ascorbate effects were consistently greater in a tissue preparation which was originally homogenized in an isotonic sucrose medium and centrifuged, and the cell debris discarded (as was done in preparation C), than in one in which the tissue was homogenized in a hypotonic medium and in which no low-speed centrifugation was done (as was done in preparation A). In other experiments, of several cations tested, only ferrous and ferric potentiated the above-described effects of ascorbate. Some ascorbic acid derivatives (e.g., isoascorbic acid) had properties similar to those of ascorbic acid, whereas several reducing agents could, in the presence of added iron salts, cause both a lipid peroxidation and a loss of [3H]spiroperidol binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced detection of H2O2 in cells expressing Horseradish Peroxidase

A new procedure for fluorescent detection of intracellular H₂O₂ in cells transiently expressing the catalyst Horseradish Peroxidase (HRP) is setup and validated. More specific reaction with HRP largely amplifies oxidation of the redox probes used (2′,7′-dichlorodihydrofluorescein and dihydrorhodamine). Expression of HRP does not affect cell viability. The procedure reveals MAO activity, a primary intracellular H₂O₂ source, in monolayers of intact transfected cells. The probes oxidation rate responds specifically to the MAO activation/inhibition. Their oxidation by MAO-derived H₂O₂ is sensitive to intracellular H₂O₂ competitors: it decreases when H₂O₂ is removed by pyruvate and it increases when the GSH-dependent removal systems are impaired. Specific response was also measured after addition of extracellular H₂O₂. Oxidation of the fluorescent probes following reaction of H₂O₂ with endogenous HRP overcomes most criticisms in their use for intracellular H₂O₂ detection. The method can be applied for direct determination in plate reader and is proposed to detect H₂O₂ generation in physio-pathological cell models.     

脂质过氧化引起的DNA损伤研究进展

脂质过氧化可以引起各种碱基损伤、DNA链断裂和各种荧光产物生成,并对DNA分子鸟嘌呤碱基具有选择性损伤.过渡金属离子可以明显加深脂质过氧化对DNA的损伤程度.多种抗氧化剂、活性氧自由基清除剂对脂质过氧化引起的DNA损伤有一定程度的保护作用.具有致突、致癌作用的8－羟基鸟嘌呤已经观察到．脂质过氧化的致突变、致癌变作用机制引起了人们的极大兴趣．     

Underestimation of D-glucose phosphorylation as measured by 3H2O production from D-[2-3H]glucose

In rat pancreatic islets, tumoral islet cells (RINm5F line), parotid gland, and in human erythrocytes, but not in rat hepatocytes, the production of 3H2O from D-[2-3H]glucose is 20-30% lower than from D-[5-3H]glucose. This coincides with the production of tritiated lactic acid from D-[2-3H]glucose and may be attributable to an intramolecular hydrogen transfer in the phosphoglucoisomerase reaction. It is concluded that the production of 3H2O from D-[2-3H]glucose is not a reliable tool to assess the total rate of hexose phosphorylation.     

Synthesis of [11-2H2], [8-2H2], [7-2H2], [6-2H2], [5-2H2], [4-2H2] and [3-2H2] cis-9-octadecenoates

Deuterated oleates have been synthesized by semihydrogenation of acetylenic intermediates. [11-²H₂]Oleate was prepared by two-carbon chain extension of the C₁₆ alcohol obtained from [1-²H₂]octyl bromide and 7-octyn-1-ol. [8-²H₂] and [7-²H₂]oleates were both prepared from dimethyl suberate, tetradeutero intermediate C₁₆ alcohols were synthesized from [1,8-²H₄] and [2,7-²H₄]octane diols by monobromination, conversion to deuterated 9-decyn-1-ols and reaction with octyl bromide. Oxidation gave [8-²H₂]-9-octadecynoate and [2,7-²H₂]-9-octadecynoate, after semihydrogenation of the latter, deuterons at C-2 were removed by exchange with aqueous alkali. [6-²H₂] and [5-²H₂]oleates were obtained from methyl 5-tetradecynoate, semihydrogenation, deuterium exchange at C-2 and two malonate extensions gave [6-²H₂]oleate; reduction with lithium aluminum deuteride, two malonate extensions and semihydrogenation gave the [5-²H₂] ester. [4-²H₂] and [3-²H₂]oleates were both obtained from methyl 7-cis-hexadecenoate, exchange of the α protons and chain extension gave the [4-²H₂] ester and reduction with lithium aluminum deuteride and chain extension gave the [3-²H₂] ester.     

Synthesis of [2H8]estradiol, [2H7]estrone, [2H6]2-hydroxyestrone and [2H6]4-hydroxyestrone as internal standards for selected ion monitoring

The synthesis of [²H₈]estradiol, [²H₇]estrone, [²H₆]2-hydroxyestrone and [²H₆]4-hydroxyestrone from estrone (as a source) is described. The high isotopical purity renders the labelled compounds as suitable carriers and internal standards for quantitative gas chromatography — mass spectrometry. The content of protonium-form (i.e. natural) estrogens in the labelled derivatives ranged from 0.12 % to 2.58 %. The perfcrmance of these compounds in quantitative assays using selected ion monitoring has been established; and this allows the determination of estrogens from biological material in the lower picogram range.     

Synthesis of methyl [18-2H3], [17-2H2], [16-2H2], [14-2H2] and [12-2H2] cis-9-octadecenoates

Methyl [17-²H₂]oleate was prepared by stepwise reduction from 17-oxooleate in 24% yield. Methyl [18-²H₃], [16-²H₂], [14-²H₂] and [12-²H₂] oleates were synthesized from appropriately deuterated octylbromides by conversion to deuterated 7-hexadecyn-1-ols and chain extention to deuterated stearolates followed by semihydrogenation; overall yields were about 17%.     

Bioinorganic study on [Fe(promethazine)2H2O Cl]2+ complex

The coordination chemistry of iron (III) is the environment of an antihistaminic drug, promethazine has been explained to include a low spin, six-coordinate complex [Fe(Prometha)2(H2O) Cl] Cl2. Metaldrug interaction in vitro in aqueous KCl phase was studied polarographically at physiological pH and temperature. On the basis of elemental, magnetic, conductometric, IR, UV-visible, NMR spectroscopic analysis it is concluded that in solid phase two promethazine molecules with their N,N donor sites encompass the metal. Mass spectral study on the complex confirms that one of the three chlorides is involved in the coordination. The respective changes in the antihistaminic activity of the drug as a result of complexation has been determined and a possible mechanism is suggested.     

The mechanism of alkyldihydroxyacetone-P synthase. Formation of [3H]H2O from acyl[1-R-3H]dihydroxyacetone-P by purified alkyldihydroxyacetone-P synthase in the absence of acylhydrolase activity

Alkyldihydroxyacetone-P (alkyl-DHAP) synthase catalyzes the exchange of the fatty acid esterified to C-1 of the DHAP portion of acyl-DHAP for a fatty alcohol to form 1-O-alkyl-DHAP, the first ether-linked intermediate in ether lipid biosynthesis. Another characteristic of the reaction is the exchange of the pro-R hydrogen at C-1. We have investigated this hydrogen exchange using palmitoyl-[1-R-3H]DHAP and a 1000-fold purified preparation of alkyl-DHAP synthase. We found a small but significant pro-R hydrogen exchange in the absence of the co-substrate, fatty alcohol. When [14C]hexadecanol was added, the increase in pro-R 3H exchange was equal to the [14C]hexadecyl-DHAP formed. Addition of [14C]palmitic acid resulted in an increase in pro-R 3H exchange that matched the formation of [14C]palmitoyl-DHAP by the acyl exchange activity of alkyl-DHAP synthase. Furthermore, although whole microsomes contain at least two acyl hydrolases for acyl-DHAP, purified preparations of alkyl-DHAP synthase do not form DHAP from acyl-DHAP. These results are discussed with respect to data obtained from other laboratories using whole microsomes and in support of our proposed ping-pong mechanism for alkyl-DHAP synthase.     

消毒剂过氧化氢脲性能的实验研究

过氧化氢脲为过氧化氢类消毒剂,实验表明,其5%水溶液25℃时有良好的的杀菌作用。杀灭细菌营养繁殖体需2min,作用15min可将细菌芽孢全部杀灭。作用30min可将HbBsAg完全灭活。提高浓度、温度,延长作用时间或降低pH值,可加强其杀菌作用。     

Chemoenzymatic synthesis of stable isotope labeled UDP-N-[2H]-acetyl-glucosamine and [2H]-acetyl-chitooligosaccharides

Labeled UDP-GlcNAc and chitooligosaccharides should be powerful tools for studies of N-acetylglucosaminyltransferase such as chitin synthases. We describe here a rapid, inexpensive and a common strategie for the chemoenzymatic synthesis of uridine 5′-diphospho-N-[²H]-acetyl-glucosamine and the chemical preparation of N-[²H]-acetyl chitooligosaccharides (from 2 to 5 mers). Deuterated UDP-GlcNAc analogue was tested as chitin synthase substrate and it exhibited an incorporation level in chitin as the natural substrate. Deuterium labeling of carbohydrates present different advantages: it is a stable isotope and allows glycosyltransferase mechanism studies by a mass spectrometry approach.     

Fe2+-Induced Lysis and Lipid Peroxidation of Chromaffin Granules

Chromaffin granules, the catecholaminergic storage granules from adrenal chromaffin cells, lysed in 10(-9)-10(-7) M Fe2+. Lysis was accompanied by the production of malondialdehyde which results from lipid peroxidation. Both chromaffin granule lysis and malondialdehyde production were inhibited by the free radical trapping agent butylated hydroxytoluene but not by catalase and/or superoxide dismutase. The results suggest that lysis resulted from a direct transfer of electrons from Fe2+ to a component of the chromaffin granule membrane without the participation of either superoxide or hydrogen peroxide and may have resulted from lipid peroxidation. In some experiments, ascorbate alone induced chromaffin granule lysis which was inhibited by EDTA, EGTA, or deferoxamine. The lysis was probably caused by trace amounts of reducible polyvalent cation. Lysis sometimes occurred when Ca2+ was added with EGTA (10 microM free Ca2+ concentration) and was consistently observed together with malondialdehyde production in the presence of Ca2+, EGTA, and 10 microM Fe2+ (total concentration). The apparent Ca2+ dependency for chromaffin granule lysis and malondialdehyde production was probably caused by a trace reducible polyvalent ion displaced by Ca2+ from EGTA and not by a Ca2+-dependent reaction involving the chromaffin granule.     

Study of dynein ATPase by oxygen isotope exchange H2(18)O in equilibrium with [16O]-Pi

The oxygen isotope exchange reactions catalyzed by sea urchin Strongylocentrotus intermedius spermatozoa dynein I were studied with a view of comparing molecular mechanisms of ATP hydrolysis by dynein and myosin ATPases. It was demonstrated that the isotope exchange takes place during ATP hydrolysis and during enzyme incubation with ADP and Pi and is absent when the enzyme is incubated with Pi. It was assumed that the molecular mechanisms of ATP hydrolysis by dynein I and myosin are identical.     

镉诱导的菊芋幼苗膜脂过氧化和抗氧化酶活性及过氧化物酶同工酶的改变

用含有不同浓度(0~400μmol/L)Cd(NO3)2的Hoagland营养液处理砂培的菊芋。处理50d后,测定植物体内镉积累量以及过氧化物酶(POD)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性,并对POD同工酶进行电泳分析。发现在Cd50~100μmol/L浓度内,随着镉浓度的升高,菊芋根和叶中镉的积累量显著增加,而随后积累量的增加有所减少。根和叶中MDA含量显著上升,说明镉引起了膜脂过氧化。0~100μmol/LCd处理,根和叶中POD活性随Cd浓度增加而增强,而在200~400μmol/LCd处理下有所减弱。根和叶SOD活性在50~200μmol/LCd处理下随Cd浓度增加而增强,而在400μmol/LCd处理下SOD活性明显受到抑制。根和叶CAT活性随Cd浓度升高而增强。电泳结果显示,POD同工酶变化明显,镉诱导出一条新酶带LP10。菊芋POD同工酶可以作为镉污染的土壤的生物指示剂。