Oxidant-mediated AA release from astrocytes involves cPLA(2) and iPLA(2)

Excessive generation of reactive oxygen species (ROS) in the central nervous system (CNS) is a leading cause of neuronal injury. Despite yet unknown mechanisms, oxidant compounds such as H₂O₂ have been shown to stimulate the release of arachidonic acid (AA) in a number of cell systems. In this study, H₂O₂ and menadione, a compound known to release H₂O₂ intracellularly, were used to examine the phospholipases A₂ (PLA₂) responsible for AA release from primary murine astrocytes. Both H₂O₂ and menadione dose-dependently stimulated AA release, and the release mediated by H₂O₂ was completely inhibited by catalase. H₂O₂ also stimulated phosphorylation of extracellular signal-regulated kinases (ERK1/2) and cytosolic phospholipase A₂ (cPLA₂). However, complete inhibition of cPLA₂ phosphorylation by U0126, an inhibitor for mitogen-activated protein kinase kinase (MEK) and GF109203x, a nonselective PKC inhibitor preferring the conventional and novel isoforms, only reduced H₂O₂-stimulated AA release by 50%. MAFP, a selective, active, site-directed, irreversible inhibitor of both cPLA₂ and the Ca²⁺-independent iPLA₂, nearly completely inhibited H₂O₂-mediated AA release; but, HELSS, a potent irreversible inhibitor of iPLA₂, only inhibited H₂O₂-mediated AA release by 40%. Along with the observation that H₂O₂-mediated AA release was only partially inhibited upon chelating intracellular Ca²⁺ by BAPTA, these results indicate the involvement of both cPLA₂ and iPLA₂ in H₂O₂-mediated AA release in murine astrocytes.     

穿心莲内酯衍生物抗H2O2诱导胰岛RIN-mβ细胞凋亡的蛋白组学研究

采用蛋白组学方法筛选穿心莲内酯衍生物(AL-1)抗过氧化氢诱导胰岛RIN-mβ细胞凋亡的差异蛋白质分子并探讨其作用分子机制.结果显示:AL-1浓度依赖性地提高H₂O₂处理的胰岛RIN-mβ细胞的存活率.经蛋白组学研究分析,成功地鉴定了18个与凋亡、应激等相关的蛋白,包括Prohibitin、Shmt2、RhoGDP-dissociationinhibitor-1、Galectin-1、Cyt b5、Hsps等;与对照组(H₂O₂)相比,处理组(AL-1+H₂O₂)中,有9个表达上调的蛋白和9个表达下调的蛋白.AL-1通过调控与细胞凋亡、应激等相关的蛋白发挥其抗H₂O₂诱导的凋亡作用.     

Phenotypic analysis of the ccp1Delta and ccp1Delta-ccp1W191F mutant strains of Saccharomyces cerevisiae indicates that cytochrome c peroxidase functions in oxidative-stress signaling

Yeast cytochrome c peroxidase (CCP) efficiently catalyzes the reduction of H₂O₂ to H₂O by ferrocytochrome c in vitro. The physiological function of CCP, a heme peroxidase that is targeted to the mitochondrial intermembrane space of Saccharomyces cerevisiae, is not known. CCP1-null-mutant cells in the W303-1B genetic background (ccp1Δ) grew as well as wild-type cells with glucose, ethanol, glycerol or lactate as carbon sources but with a shorter initial doubling time. Monitoring growth over 10 days demonstrated that CCP1 does not enhance mitochondrial function in unstressed cells. No role for CCP1 was apparent in cells exposed to heat stress under aerobic or anaerobic conditions. However, the detoxification function of CCP protected respiring mitochondria when cells were challenged with H₂O₂. Transformation of ccp1Δ with ccp1^W191F, which encodes the CCP^W191F mutant enzyme lacking CCP activity, significantly increased the sensitivity to H₂O₂ of exponential-phase fermenting cells. In contrast, stationary-phase (7-day) ccp1Δ-ccp1^W191F exhibited wild-type tolerance to H₂O₂, which exceeded that of ccp1Δ. Challenge with H₂O₂ caused increased CCP, superoxide dismutase and catalase antioxidant enzyme activities (but not glutathione reductase activity) in exponentially growing cells and decreased antioxidant activities in stationary-phase cells. Although unstressed stationary-phase ccp1Δ exhibited the highest catalase and glutathione reductase activities, a greater loss of these antioxidant activities was observed on H₂O₂ exposure in ccp1Δ than in ccp1Δ-ccp1^W191F and wild-type cells. The phenotypic differences reported here between the ccp1Δ and ccp1Δ-ccp1^W191F strains lacking CCP activity provide strong evidence that CCP has separate antioxidant and signaling functions in yeast.     

Ca2+参与H2O2促进盐碱混合胁迫下燕麦种子的萌发和成苗

盐碱胁迫是制约作物高产优质的重要因素,Ca²⁺和H₂O₂作为信号分子参与作物逆境响应调节。为了解Ca²⁺是否参与H₂O₂对盐碱胁迫下植物种子萌发和成苗的调控,以燕麦（Avena nude）为试验材料,采用隶属函数分析方法,研究了胞外游离Ca²⁺螯合剂EGTA、质膜Ca²⁺通道阻断剂LaCl₃和液泡膜Ca²⁺释放抑制剂钌红（RR）与H₂O₂共处理对盐碱混合（NaCl：Na₂SO₄：NaHCO₃：Na₂CO₃=12：8：9：1）胁迫下种子萌发和成苗的影响。结果表明,25~200 mmol·L^-1盐碱混合胁迫显著抑制燕麦的种子萌发和成苗,抑制程度随浓度提高而增强;0.001~2 mmol·L^-1 H₂O₂能够促进燕麦种子的萌发和成苗,且0.5 mmol·L^-1 H₂O₂可以显著缓解75 mmol·L^-1盐碱混合胁迫对燕麦种子萌发和成苗的抑制作用;而EGTA、LaCl₃和RR均能消减H₂O₂对盐碱混合胁迫下燕麦种子萌发和成苗的促进作用。表明Ca²⁺参与H₂O₂促进盐碱混合胁迫下燕麦种子萌发和成苗的信号转导过程。     

油桃花芽破眠过程中H2O2代谢与Ca2+转运的关系

利用化学测定法分析高温、单氰胺和TDZ 3种破眠处理对“曙光”油桃休眠花芽H₂O₂代谢的主要影响,利用非损伤微测技术检测H₂O₂对休眠芽Ca²⁺转运的影响,研究H₂O₂在芽休眠解除过程中的调控作用.结果表明： 在深休眠时期,高温和单氰胺处理均能诱导芽内H₂O₂含量升高和过氧化氢酶(CAT)活性降低,并具有显著的破眠作用;TDZ对H₂O₂含量及CAT、过氧化物酶(POD)活性影响不大,破眠效果较差.休眠花芽原基组织钙通道活跃,对外源Ca²⁺呈吸收状态.外源H₂O₂可诱导休眠花芽原基组织Ca²⁺转运发生变化,低浓度H₂O₂降低Ca²⁺吸收速率,高浓度H₂O₂使组织对Ca²⁺的转运由吸收转变为释放.这表明休眠芽内H₂O₂信号和Ca²⁺信号相关联,通过诱导H₂O₂积累调控Ca²⁺信号可能在高温和单氰胺打破休眠的信号转导过程中起重要作用.     

Does hydrogen peroxide exist “free” in biological systems?

Hydrogen peroxide (H₂O₂) can diffuse far from the site of production to intracellular locations where biological effects may be greater. The diffusion range is extended by H₂O₂ carriers formed spontaneously by hydrogen bonding with monomeric and polymeric compounds, including amino and dicarboxylic acids, peptides, proteins, nucleic acid bases, and nucleosides. Hydrogen peroxide adducts (HPAs) are readily synthesized, e.g., crystalline histidine (His)-H₂O₂ adducts. An equilibrium exists between an adduct-forming compound and H₂O₂. The detection and relative stabilities of HPAs are measured by the degree of decomposition of H₂O₂ as influenced by test compounds in buffered solution competing with glucose or fructose for H₂O₂. The HPAs delay decomposition of H₂O₂ up to several hundredfold. The overall charge on an HPA, i.e., its ability to penetrate cell membranes, influences the cytotoxic and clastogenic effects of H₂O₂. Growth inhibition of Salmonella typhimurium LT₂ by H₂O₂ is enhanced by neutral HPAs but decreased by anionic HPAs. Addition of catalase 1, 10, or 30 min after inoculation of S. typhimurium LT₂ reduces or nearly eliminates partial growth inhibition by H₂O₂, but a neutral HPA, expecially his-H₂O₂, transported H₂O₂ into the cells within 1 min, and in about 10 min completely inhibited growth. The stability of HPAs decreases with increasing pH or increasing temperature, while added Fe(II) in the presence and absence of EDTA accelerates H₂O₂ and HPA decomposition. Calculations indicate H₂O₂ hydrogen bonds with nucleic acid-base pairs with no apparent bond strain and energy stabilization comparable to normal hydrogen bonding.     

Turning point in apoptosis/necrosis induced by hydrogen peroxide

The turning point between apoptosis and necrosis induced by hydrogen peroxide (H₂O₂) have been investigated using human T-lymphoma Jurkat cells. Cells treated with 50 μM H₂O₂ exhibited caspase-9 and caspase-3 activation, finally leading to apoptotic cell death. Treatment with 500 μM H₂O₂ did not exhibit caspase activation and changed the mode of death to necrosis. On the other hand, the release of cytochrome c from the mitochondria was observed under both conditions. Treatment with 500 μM H₂O₂, but not with 50 μM H₂O₂, caused a marked decrease in the intracellular ATP level; this is essential for apoptosome formation. H₂O₂-reducing enzymes such as cellular glutathione peroxidase (cGPx) and catalase, which are important for the activation of caspases, were active under the 500 μM H₂O₂ condition. Prevention of intracellular ATP loss, which did not influence cytochrome c release, significantly activated caspases, changing the mode of cell death from necrosis to apoptosis. These results suggest that ATP-dependent apoptosome formation determines whether H₂O₂-induced cell death is due to apoptosis or necrosis.     

Influence of chelators and iron ions on the production and degradation of H2O2 by beta-amyloid-copper complexes

β-Amyloid peptide (Aβ) 1–42, involved in the pathogenesis of Alzheimer’s disease, binds copper ions to form Aβ · Cu_n complexes that are able to generate H₂O₂ in the presence of a reductant and O₂. The production of H₂O₂ can be stopped with chelators. More reactive than H₂O₂ itself, hydroxyl radicals HO (generated when a reduced redox active metal complex interacts with H₂O₂) are also probably involved in the oxidative stress that creates brain damage during the disease. We report in the present work a method to monitor the effect of chelating agents on the production of hydrogen peroxide by metallo-amyloid peptides. The addition of H₂O₂ associated to a pre-incubation step between ascorbate and Aβ · Cu_n allows to study the formation of H₂O₂ but also, at the same time, its transformation by the copper complexes. Aβ · Cu_n peptides produce but do not efficiently degrade H₂O₂. The reported analytic method, associated to precipitation experiments of copper-containing amyloid peptides, allows to study the inhibition of H₂O₂ production by chelators. The action of a ligand such as EDTA is probably due to the removal of the copper ions from Aβ · Cu_n, whereas bidentate ligands such as 8-hydroxyquinolines probably act via the formation of ternary complexes with Aβ · Cu_n. The redox activity of these bidentate ligands can be modulated by the incorporation or the modification of substituents on the quinoline heterocycle.     

胞外H2O2及NADPH氧化酶参与了铜胁迫对植物细胞死亡的诱导

以烟草悬浮细胞BY-2(Nicotiana tabacum L.cv.Bright Yellow-2)为材料,探讨了在铜离子胁迫下植物细胞死亡发生过程中胞外H₂O₂及NADPH氧化酶所扮演的角色。实验结果表明,随着外源CuCl₂浓度的上升(从0~700 μmol·L^-1),细胞死亡水平不断上升,且胞外H₂O₂的水平也不断增加。在300 μmol·L^-1的CuCl₂诱导细胞死亡的过程中,加入H₂O₂清除剂N-N-二甲基硫脲(DMTU)降低了胞外CuCl₂胁迫下H₂O₂含量增加的同时也降低了细胞死亡水平的上升,这一观察表明了铜离子胁迫所导致的细胞死亡的发生和胞外H₂O₂的增加有关。进一步的研究表明,300 μmol·L^-1 CuCl₂的胁迫导致了NADPH氧化酶活性的显著性上升,而加入NADPH氧化酶的抑制剂（二亚苯基碘,DPI,)则降低了CuCl₂胁迫所导致的细胞死亡和胞外H₂O₂含量的上升。上述结果表明,胞外H₂O₂和NADPH氧化酶参与了CuCl₂对植物细胞死亡的诱导作用。     

外源H2O2对盐碱混合胁迫下裸燕麦幼苗生长和抗性生理的影响

为探讨信号分子过氧化氢（H₂O₂）增强裸燕麦盐碱耐性的作用及其生理机制,以裸燕麦品种‘定莜6号’为材料,在日光温室内用珍珠岩培养幼苗至三叶一心期时叶面喷施0.01 mmol·L^-1 H₂O₂的同时根部浇灌75 mmol·L^-1盐碱混合溶液（NaCl：Na₂SO₄：NaHCO₃：Na₂CO₃=12：8：9：1）或添加H₂O₂淬灭剂二甲基硫脲（DMTU）,研究对幼苗生长及叶片光合色素含量、活性氧代谢和渗透调节物质积累的影响。结果表明：喷施H₂O₂能够缓解盐碱混合胁迫对裸燕麦幼苗生长的抑制,提高幼苗根长、株高和植株干重及叶片叶绿素a、叶绿素b、总叶绿素、类胡萝卜素含量和超氧化物歧化酶、过氧化物酶、过氧化氢酶、抗坏血酸过氧化物酶活性,降低超氧阴离子、H₂O₂、丙二醛、抗坏血酸、谷胱甘肽和游离氨基酸含量,促进抗氧化物质类黄酮、总酚和原花青素及渗透调节物质可溶性蛋白质、可溶性糖和脯氨酸积累。添加DMTU部分或完全逆转了H₂O₂的上述作用。采用隶属函数综合评价显示,喷施H₂O₂提高了盐碱混合胁迫下裸燕麦幼苗的综合评价值D,添加DMTU完全逆转了H₂O₂对D值的提升作用。表明外源H₂O₂通过参与活性氧代谢和渗透调节物质积累等生理代谢调控缓解盐碱混合胁迫诱导的氧化伤害和生长抑制,从而提高裸燕麦对盐碱胁迫的适应能力。     

Aging, cytochrome oxidase activity, and hydrogen peroxide release by mitochondria

The objective of this study was to explore the possible cause(s) underlying the previously observed, age-related increase in the rate of mitochondrial H₂O₂ release in the housefly. The hypothesis that an imbalance between different respiratory complexes may be a causal factor was tested. Cytochrome c oxidase activity was found to sharply decline in the latter part of the life span of the flies. Effects of different substrates and respiratory inhibitors were determined in order to ascertain if a decrease in cytochrome c oxidase activity could be responsible for the increased H₂O₂ release. H₂O₂ was measured spectrofluorometrically using horseradish peroxidase and p-hydrophenylacetate as an indicator. Neither NADH-linked substrates nor succinate caused a stimulation of H₂O₂ production. H₂O₂ release by mitochondria, inhibited with rotenone and antimycin A, was greatly increased upon supplementation with -glycerophosphate; however, the further addition of KCN or myxothiazol, to such preparations, caused a depression of H₂O₂ generation. In contrast, relatively low concentrations of KCN or myxothiazol were found to stimulate H₂O₂ release in insect mitochondria supplemented with -glycerophosphate and exposed to rotenone, but not antimycin A. Results are interpreted to suggest that partial inhibition of cytochrome c oxidase activity can lead to the stimulation of mictochondrial H₂O₂ production in the housefly at site(s) other than NADH dehydrogenase and ubisemiquinone/ cytochrome b region; a possible source may be glycerophosphate dehydrogenase.     

不同氧浓度下C2C12细胞的Nrf2抗氧化系统对H2O2刺激的反应*

目的:探讨不同氧浓度下小鼠骨骼肌卫星细胞系(C2C12细胞)对H₂O₂刺激反应的变化及其机制。方法:小鼠骨骼肌卫星细胞系(C2C12细胞),经培养复苏后,将细胞分为7组,每组设8个复孔,各组分别加入浓度为0.1 mmol/L、0.25 mmol/L、0.5 mmol/L、0.75 mmol/L、1 mmol/L、2 mmol/L的H₂O₂,分别作用1 h、2 h后测细胞活力,选择细胞H₂O₂刺激的最佳作用时间和浓度;C2C12细胞分为不同氧浓度组:21% O₂、12% O₂、8% O₂、5% O₂每组设8个复孔,12 h后,H₂O₂作用1 h,收集细胞;检测细胞Nrf2蛋白荧光和蛋白表达量,测定Nrf2和抗氧化酶SOD1、SOD2、CAT、NQO-1、HO-1、GPX-1 的mRNA表达量及细胞ROS水平。结果:选择H₂O₂作用时间相对较短的1 h和浓度0.5 mmol/L作为本实验的H₂O₂刺激条件。与21%O₂组相比,12%O₂组细胞Nrf2蛋白荧光增强,Nrf2 的mRNA和蛋白表达以及抗氧化酶SOD1、SOD2、CAT、NQO-1、HO-1、GPX-1的 mRNA表达均显著增加(P＜0.05或P＜0.01),细胞 ROS水平明显降低(P＜0.01);8%O₂组仅GPX-1 mRNA显著增加(P＜0.05),其他指标变化不大;5%O₂组细胞 Nrf2 mRNA和蛋白表达以及抗氧化酶SOD1、SOD2、NQO-1、GPX-1的 mRNA表达均明显降低(P＜0.05或P＜0.01),细胞 ROS水平则明显升高(P＜0.01)。结论:不同氧浓度下C2C12细胞中Nrf2介导的抗氧化系统对H₂O₂刺激反应不同,12 h的12% O₂浓度可促进C2C12细胞Nrf2的抗氧化作用,而5% O₂浓度的严重低氧则作用相反。     

Non-oxygen-forming pathways of hydrogen peroxide degradation by bovine liver catalase at low hydrogen peroxide fluxes

Heme catalases are considered to degrade two molecules of H₂O₂ to two molecules of H₂O and one molecule of O₂ employing the catalatic cycle. We here studied the catalytic behaviour of bovine liver catalase at low fluxes of H₂O₂ (relative to catalase concentration), adjusted by H₂O₂-generating systems. At a ratio of a H₂O₂ flux (given in μM/min^- 1) to catalase concentration (given in μM) of 10 min^- 1 and above, H₂O₂ degradation occurred via the catalatic cycle. At lower ratios, however, H₂O₂ degradation proceeded with increasingly diminished production of O₂. At a ratio of 1 min^- 1, O₂ formation could no longer be observed, although the enzyme still degraded H₂O₂. These results strongly suggest that at low physiological H₂O₂ fluxes H₂O₂ is preferentially metabolised reductively to H₂O, without release of O₂. The pathways involved in the reductive metabolism of H₂O₂ are presumably those previously reported as inactivation and reactivation pathways. They start from compound I and are operative at low and high H₂O₂ fluxes but kinetically outcompete the reaction of compound I with H₂O₂ at low H₂O₂ production rates. In the absence of NADPH, the reducing equivalents for the reductive metabolism of H₂O₂ are most likely provided by the protein moiety of the enzyme. In the presence of NADPH, they are at least in part provided by the coenzyme.     

黑腹果蝇white基因在H2O2影响下卷翅形成中的作用及机理

卷翅是果蝇遗传学上最常用的标记之一,但卷翅形成的具体机制还不清楚.过去的研究发现,理化刺激影响果蝇卷翅的形成.我们最近研究发现,H₂O₂处理不仅会影响果蝇的羽化率,还会使其出现卷翅现象.本研究通过改变H₂O₂浓度、果蝇培养温度和H₂O₂处理时间,探讨影响黑腹果蝇卷翅形成的具体因素,并对其超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-PX)活力进行检测,探讨H₂O₂对果蝇抗氧化能力的影响.结果表明: 果蝇的羽化率与H₂O₂浓度成反比.温度、H₂O₂浓度和H₂O₂处理时间的改变均会影响果蝇翅的卷曲程度和卷翅果蝇所占的比例.其中white基因突变果蝇对这3种条件反应最明显,mini-white(white基因回复突变)果蝇却可以拯救该表型,它的反应与野生型OR相似.H₂O₂对含Cy基因的果蝇卷翅的形成也有一定的影响,可以加大果蝇翅的卷曲程度.对SOD、CAT和GSH-PX活力检测发现,H₂O₂处理会使果蝇的抗氧化能力降低.实时荧光定量PCR检测发现,H₂O₂处理会导致果蝇基因表达量发生改变.黑腹果蝇卷翅形成是一个十分复杂的过程,H₂O₂可能作为某种信号分子或是间接影响某种因子参与黑腹果蝇的卷翅形成过程.该卷翅形成过程可能与Cy基因导致的果蝇卷翅过程是同一个信号途径,两者也可能是通过不同的模式进行调控的.     

Cocoa procyanidins protect PC12 cells from hydrogen-peroxide-induced apoptosis by inhibiting activation of p38 MAPK and JNK

Oxidative stress induced by reactive oxygen species has been strongly associated with the pathogenesis of neurodegenerative disorders, including Alzheimer's disease. In this study, we investigated the possible protective effects of a cocoa procyanidin fraction (CPF) and procyanidin B2 (epicatechin-(4β-8)-epicatechin) – a major polyphenol in cocoa – against apoptosis of PC12 rat pheochromocytoma (PC12) cells induced by hydrogen peroxide (H₂O₂). CPF (1 and 5 μg/ml) and procyanidin B2 (1 and 5 μM) reduced PC12 cell death caused by H₂O₂, as determined by MTT and trypan blue exclusion assays. CPF and procyanidin B2 attenuated the H₂O₂-induced fragmentation of nucleus and DNA in PC12 cells. Western blot data demonstrated that H₂O₂ induced cleavage of poly(ADP-ribose)polymerase (PARP), downregulated Bcl-X_L and Bcl-2 in PC12 cells. Pretreatment with CPF or procyanidin B2 before H₂O₂ treatment diminished PARP cleavage and increased Bcl-X_L and Bcl-2 expression compared with those only treated with H₂O₂. Activation of caspase-3 by H₂O₂ was inhibited by pretreatment with CPF or procyanidin B2. Furthermore, H₂O₂-induced rapid and significant phosphorylation of c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and both of these effects were attenuated by CPF or procyanidin B2 treatment. These results suggest that the protective effects of CPF and procyanidin B2 against H₂O₂-induced apoptosis involve inhibiting the downregulation of Bcl-X_L and Bcl-2 expression through blocking the activation of JNK and p38 MAPK.     

低浓度过氧化氢对HaCat细胞增殖和大鼠创面愈合的作用与机制研究

目的 探讨低浓度过氧化氢(H2O2)对创面愈合的促进作用及其可能的作用机制.方法 建立大鼠全层皮肤缺损创面模型,将大鼠分为对照组(使用生理盐水)、高、低浓度H2O2组(分别使用3％、0.01％H2O2干预).选取第0、3、6、9、12、15、18天共7个时间点评估创面愈合率,并在第3、6、9天对创面组织样本进行组织病理...     

Involvement of protein kinase C delta in the alteration of mitochondrial mass in human cells under oxidative stress

Alteration of mitochondrial mass of human 143B osteosarcoma cells upon exposure to hydrogen peroxide (H₂O₂) was investigated. We found that mitochondrial mass and the intracellular level of H₂O₂ were increased by exogenous H₂O₂, which was accompanied with up-regulation of functional PKCδ. To investigate the role of PKCδ in H₂O₂-induced increase of mitochondrial mass, we treated 143B cells with PKCδ activator, bistratene A, and PKCδ inhibitor, rottlerin, respectively. The results show that bistratene A caused an increase of mitochondrial mass and that the H₂O₂-induced increase of mitochondrial mass was completely suppressed by rottlerin. Furthermore, we found that activation of PKCδ by bistratene A increased the intracellular levels of H₂O₂ and MnSOD protein expression. By contrast, suppression of PKCδ by rottlerin decreased the intracellular levels of H₂O₂ and MnSOD protein expression. Moreover, we noted that MnSOD expression was highly correlated with the expression of p53, which was controlled by PKCδ. Finally, we demonstrated that PKCδ was overexpressed in skin fibroblasts of patients with MERRF syndrome. Taken together, we conclude that PKCδ is involved in the regulation of mitochondrial mass and intracellular H₂O₂ in human cells and may play a key role in the overproliferation of mitochondria in the affected tissues of patients with mitochondrial diseases such as MERRF syndrome.     

Iron Release from Metmyoglobin, Methaemoglobin and Cytochrome C by A System Generating Hydrogen Peroxide

The reaction of H₂O₂ with resting metmyoglobin (MetMb), methaemoglobin (MetHb) and cytochrome-c (Cyt-c) was studied in the Soret and visible regions. The differences between the original and the final peak heights of the native haemproteins at 408 nm was found to be directly proportional to the loss of iron from the molecule. The release of iron from haemproteins was studied in a system generating H₂O₂ continuously at a low rate by an enzymic system, or by addition of large amounts of H₂O₂. Cytochrome-c, methaemoglobin and metmyoglobin during interaction with H₂O₂ at a concentration of 200 μM release 40%, 20% and 3%, respectively, of molecular iron after l0min. The inhibition of haem degradation and iron release by enzymatically-generated H₂O₂ was determined using several hydroxyl radical scavengers, reducing agents and antioxienzymes, such as superoxide dismutase, catalase and caeruloplasmin.     

Ascorbate and H2O2 induced oxidative DNA damage in Jurkat cells

The effect of vitamin C (ascorbate) on oxidative DNA damage was examined by first incubating cells with dehydroascorbate, which boosts the intracellular concentration of ascorbate, and then exposing cells to H₂O₂. Oxidative DNA damage was estimated by the analysis of 5-hydroxy-2′-deoxycytidine (oh⁵dCyd) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (oxo⁸dGuo). The presence of a high concentration of ascorbate (30 mM), compared to the absence of ascorbate in cells, when exposed to H₂O₂ (200 μM), resulted in a remarkable sensitization of oh⁵dCyd from 2.7 ± 0.6 to 40.8 ± 6.1 lesions /10⁶ dCyd (15-fold). In contrast, the level of oxo⁸dGuo increased from 8.4 ± 0.4 to 12.1 ± 0.5 lesions/10⁶ dGuo (50%). The formation of oh⁵dCyd was also observed at lower concentrations of intracellular ascorbate and exogenous H₂O₂. Additional studies showed that replacement of H₂O₂ with tert-butyl hydroperoxide completely abolished damage, and that preincubation with iron and desferroxamine increased and decreased this damage, respectively. The latter studies suggest that a Fenton reaction is involved in the mechanism of damage. In conclusion, we report a novel model system in which ascorbate sensitizes H₂O₂-induced oxidative DNA damage in cells, leading to elevated levels of oh⁵dCyd and oxo⁸dGuo, with a strong bias toward the formation of oh⁵dCyd.