Hydrogen Peroxide and the Proliferation of Bhk-21 Cells

Intracellular levels of H2O2 in BHK-21 cells are not static but decline progressively with cell growth. Exposure of cells to inhibitors of catalase, or glutathione peroxidase, not only diminishes this decline but also depresses rates of cell proliferation, suggesting important growth regulatory roles for those antioxidant enzymes. Other agents which also diminish the growth-associated decline in intracellular levels of H₂O₂, such as the superoxide dismutase mimic, copper II—(3,5-diisopropylsalicylate)₂, or docosahexaenoic acid, also reduced cell proliferation. In contrast, proliferation can be stimulated by the addition of 1 μM exogenous H₂O₂ to the culture medium. Under these conditions, however, intracellular levels of H₂O₂ are unaffected, whereas there is a reduction in intracellular levels of glutathione. It is argued that critical balances between intracellular levels of both H₂O₂ and glutathione are of significance in relation both to growth stimulation and inhibition. In addition growth stimulatory concentrations of H₂O₂, whilst initially leading to increased intracellular levels of lipid peroxidation breakdown products, appear to “trigger” their metabolism, possibly through aldehyde dehydrogenase, whose activity is also stimulated by H₂O₂     

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。     

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.     

硫化氢对低温胁迫下甜樱桃柱头和子房线粒体功能的影响

为探索低温胁迫下外源硫化氢(H₂S)对甜樱桃花的柱头和子房线粒体功能的影响,本研究以甜樱桃品种‘早大果’花枝为试材,在-2 ℃低温下喷施0.05 mmol·L^-1硫氢化钠(NaHS,H₂S供体)和15 μmmol·L^-1 次牛磺酸(HT、H₂S清除剂),测定柱头和子房线粒体中活性氧、抗氧化酶和线粒体膜通透性转换孔(MPTP)开放程度、膜流动性、膜电位和细胞色素(Cyt c/a)比值变化。结果表明: 低温胁迫导致线粒体内过氧化氢(H₂O₂)和丙二醛(MDA)含量显著增加,线粒体MPTP明显增大,膜流动性降低,膜电位和线粒体Cyt c/a吸光度比值、膜H⁺-ATPase活性显著下降,线粒体结构受到损伤。低温胁迫下,外施0.05 mmol·L^-1 NaHS可显著降低低温胁迫下柱头和子房线粒体H₂O₂和MDA含量,在较长时间内维持较高的超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性,减小线粒体MPTP开放程度,增强线粒体膜流动性,提高线粒体膜电位、Cyt c/a值和膜H⁺-ATPase活性;NaHS清除剂HT则抵消NaHS对上述参数的影响。综上所述,外源H₂S可以提高低温胁迫下甜樱桃柱头和子房线粒体抗氧化酶活性,减少H₂O₂和MDA积累,提高膜H⁺-ATPase活性,稳定线粒体膜结构和功能,进而缓解低温胁迫对花器官的伤害。     

Triphlorethol-A from Ecklonia cava protects V79-4 lung fibroblast against hydrogen peroxide induced cell damage

In the present study, triphlorethol-A, a phlorotannin, was isolated from Ecklonia cava and its antioxidant properties were investigated. Triphlorethol-A was found to scavenge intracellular reactive oxygen species (ROS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, and thus prevented lipid peroxidation. The radical scavenging activity of triphlorethol-A protected the Chinese hamster lung fibroblast (V79-4) cells exposed to hydrogen peroxide (H₂O₂) against cell death, via the activation of ERK protein. Furthermore, triphlorethol-A reduced the apoptotic cells formation induced by H₂O₂. Triphlorethol-A increased the activities of cellular antioxidant enzymes like, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Hence, from the present study, it is suggestive that triphlorethol-A protects V79-4 cells against H₂O₂ damage by enhancing the cellular antioxidative activity.     

Hydrogen peroxide is necessary for abscisic acid-induced senescence of rice leaves

The role of H₂O₂ in abscisic acid (ABA)-induced rice leaf senescence is investigated. ABA treatment resulted in H₂O₂ production in rice leaves, which preceded the occurrence of leaf senescence. Dimethylthiourea, a chemical trap for H₂O₂, was observed to be effective in inhibiting ABA-induced senescence, ABA-increased malondialdehyde (MDA) content, ABA-increased antioxidative enzyme activities (superoxide dismutase, ascorbate peroxidase, glutathione reductase and catalase), and ABA-decreased antioxidant contents (ascorbic acid and reduced glutathione) in rice leaves. Diphenyleneiodonium chloride (DPI) and imidazole (IMD), inhibitors of NADPH oxidase, and KCN and NaN₃, inhibitors of peroxidase, prevented ABA-induced H₂O₂ production, suggesting NADPH oxidase and peroxidase are H₂O₂-generating enzymes in ABA-treated rice leaves. DPI, IMD, KCN, and NaN₃ also inhibited ABA-promoted senescence, ABA-increased MDA contents, ABA-increased antioxidative enzyme activities, and ABA-decreased antioxidants in rice leaves. These results suggest that H₂O₂ is involved in ABA-induced senescence of rice leaves.     

24-表油菜素内酯对干旱胁迫下辣椒幼苗叶片抗氧化酶系统及耐旱相关基因表达的影响

报道了干旱胁迫下外源24-表油菜素内酯（EBR）对辣椒幼苗叶片H₂O₂和MDA含量,抗氧化酶活性,以及耐旱相关基因表达的影响。结果表明,0.1 μmol·L^-1 EBR处理诱导了辣椒幼苗叶片H₂O₂含量的增加,并提高了SOD、APX、CAT、DHAR、MDAR和GR活性;干旱胁迫下,EBR处理显著诱导了辣椒叶片抗氧化酶活性的增加,并抑制了H₂O₂和MDA含量的上升;EBR处理也促进了cAPX和MDAR等抗氧化酶基因的表达,以及WRKY3、WRKY6和MYB等转录因子的表达。由此认为,适宜浓度的外源EBR可能是通过信号分子H₂O₂调控辣椒叶片中WRKY和MYB等转录因子的表达以调控相关耐旱基因表达,增强细胞的抗氧化酶活性,减轻干旱造成的膜质过氧化伤害,从而增强了辣椒幼苗的耐旱性。     

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.     

Role of antioxidants in protecting cellular DNA from damage by oxidative stress

We have previously derived 2 V79 clones resistant to menadione (Md1 cells) and cadmium (Cd1 cells), respectively. They both were shown to be cross-resistant to hydrogen peroxide. There was a modification in the antioxidant repertoire in these cells as compared to the parental cells. Md1 presented an increase in catalase and glutathione peroxidase activities whereas Cd1 cells exhibited an increase in metallothionein and glutathione contents. The susceptibility of the DNA of these cells to the damaging effect of H₂O₂ was tested using the DNA precipitation assay. Both Md1 and Cd1 DNAs were more resistant to the peroxide action. In the case of Md1 cells it seems clear that the extra resistance is provided by the increase in the two H₂O₂ scavenger enzymes, catalase and glutathione peroxidase. In the case of Cd1 cells the activities of these enzymes as well as of superoxide dismutases (Cu/Zn and Mn) are unaltered as compared to the parental cells. The facts that parental cells exposed to 100 μM Zn²⁺ in the medium exhibit an increase in metallothionein but not in glutathione and that these cells become more resistant to the DNA-damaging effect of H₂O₂ suggest that this protein might play a protective role in vivo against the OH radical attack on DNA.     

Inactivation of Yeast Glutathione Reductase by Fenton Systems: Effect of Metal Chelators, Catecholamines and Thiol Compounds

Oxygen radical generating systems, namely, Cu(II)/ H₂O₂, Cu(II)/ascorbate, Cu(II)/NAD(P)H, Cu(II)/ H₂O₂/catecholamine and Cu(II)/H₂O₂/SH-compounds irreversibly inhibited yeast glutathione reductase (GR) but Cu(II)/H₂O₂ enhanced the enzyme diaphorase activity. The time course of GR inactivation by Cu(II)/H₂O₂ depended on Cu(II) and H₂O₂ concentrations and was relatively slow, as compared with the effect of Cu(II)/ascorbate. The fluorescence of the enzyme Tyr and Trp residues was modified as a result of oxidative damage. Copper chelators, catalase, bovine serum albumin and HO^˙ scavengers prevented GR inactivation by Cu(II)/H₂O₂ and related systems. Cysteine, N-acetylcysteine, N-(2-dimercaptopropi-onylglycine and penicillamine enhanced the effect of Cu(II)/H₂O₂ in a concentration- and time-dependent manner. GSH, Captopril, dihydrolipoic acid and dithiotreitol also enhanced the Cu(II)/H₂O₂ effect, their actions involving the simultaneous operation of pro-oxidant and antioxidant reactions. GSSG and try-panothione disulfide effectively protected GR against Cu(II)/H₂O₂ inactivation. Thiol compounds prevented GR inactivation by the radical cation ABTS*⁺. GR inactivation by the systems assayed correlated with their capability for HO* radical generation. The role of amino acid residues at GR active site as targets for oxygen radicals is discussed.     

Effect of coenzyme Q10 intake on endogenous coenzyme Q content, mitochondrial electron transport chain, antioxidative defenses, and life span of mice

The main purpose of this study was to determine whether intake of coenzyme Q10, which can potentially act as both an antioxidant and a prooxidant, has an impact on indicators of oxidative stress and the aging process. Mice were fed diets providing daily supplements of 0, 93, or 371 mg CoQ10 /kg body weight, starting at 3.5 months of age. Effects on mitochondrial superoxide generation, activities of oxidoreductases, protein oxidative damage, glutathione redox state, and life span of male mice were determined. Amounts of CoQ9 and CoQ10, measured after 3.5 or 17.5 months of intake, in homogenates and mitochondria of liver, heart, kidney, skeletal muscle, and brain increased with the dosage and duration of CoQ10 intake in all the tissues except brain. Activities of mitochondrial electron transport chain oxidoreductases, rates of mitochondrial O2-* generation, state 3 respiration, carbonyl content, glutathione redox state of tissues, and activities of superoxide dismutase, catalase, and glutathione peroxidase, determined at 19 or 25 months of age, were unaffected by CoQ10 administration. Life span studies, conducted on 50 mice in each group, showed that CoQ10 administration had no effect on mortality. Altogether, the results indicated that contrary to the historical view, supplemental intake of CoQ10 elevates the endogenous content of both CoQ9 and CoQ10, but has no discernable effect on the main antioxidant defenses or prooxidant generation in most tissues, and has no impact on the life span of mice.     

The role of H2O2 as a mediator of UVB-induced apoptosis in keratinocytes

Apoptosis is an active form of cell death that is initiated by a variety of stimuli, including reactive oxygen species (ROS) and ultraviolet (UV) radiation. Previously, it has been reported that UVB-irradiation of keratinocytes leads to intracellular generation of hydrogen peroxide (H₂O₂) and that antioxidants can inhibit ROS-induced apoptosis. Although both UVB-irradiation and H₂O₂-incubation led to increased intracellular H₂O₂ levels, the antioxidants catalase and glutathione monoester (GME), inhibited apoptosis only when induced by H₂O₂, not by UVB. Furthermore, extracellular signal-regulated kinase (ERK), a prominent member of the mitogen-activated protein kinase (MAPK) family, was found to be activated by treatment with both UVB and H₂O₂. Inhibition of ERK phosphorylation by pre-treatment with PD98059 resulted in enhanced apoptosis after H₂O₂-exposure. However, no significant difference of apoptosis was observed between cells with and without inhibitor pre-treatment upon UVB-irradiation. DNA damage in the form of cyclobutane pyrimidine dimers was observed after exposure to UVB, but no photoproducts were found in H₂O₂-treated cells. These results suggest a ROS-independent pathway of UVB-induced apoptosis. Although UVB-irradiation causes moderate increase in H₂O₂, the generation of H₂O₂ does not contribute to the induction of apoptosis. Moreover, activation of ERK only blocks H₂O₂-dependent apoptosis but has no impact on UVB-induced apoptosis.     

Effects of methyl viologen on the antioxidant system in cultured Salvia miltiorrhiza cells

为了探讨甲基紫精(MV)对丹参(Salvia miltiorrhiza)体内抗氧化防护系统的影响及其生理机制。以MV为诱导剂, 以敌草隆(DCMU)为抑制剂, 考察了MV与DCMU处理后丹参悬浮培养细胞中H₂O₂、丙二醛、还原型谷胱甘肽的含量以及抗氧化防护酶(超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT))活性变化和同工酶的表达差异。结果表明, MV处理显著提高了丹参培养细胞内H₂O₂、丙二醛以及还原型谷胱甘肽含量; MV处理使CAT、POD活性增强, 谱带颜色更亮, 条带增加。DCMU处理显著抑制了MV诱导的H₂O₂、丙二醛、还原型谷胱甘肽含量的增加, 抗氧化酶活性的升高和同工酶的表达。以上结果说明, MV可诱导丹参培养细胞叶绿体产生H₂O₂, H₂O₂激活了丹参培养细胞抗氧化防护系统以维持细胞正常的生理活动。     

Electron transport chain of Saccharomyces cerevisiae mitochondria is inhibited by H2O2 at succinate-cytochrome c oxidoreductase level without lipid peroxidation involvement

The deleterious effects of H₂O₂ on the electron transport chain of yeast mitochondria and on mitochondrial lipid peroxidation were evaluated. Exposure to H₂O₂ resulted in inhibition of the oxygen consumption in the uncoupled and phosphorylating states to 69% and 65%, respectively. The effect of H₂O₂ on the respiratory rate was associated with an inhibition of succinate-ubiquinone and succinate-DCIP oxidoreductase activities. Inhibitory effect of H₂O₂ on respiratory complexes was almost completely recovered by β-mercaptoethanol treatment. H₂O₂ treatment resulted in full resistance to Q_O site inhibitor myxothiazol and thus it is suggested that the quinol oxidase site (Q_O) of complex III is the target for H₂O₂. H₂O₂ did not modify basal levels of lipid peroxidation in yeast mitochondria. However, H₂O₂ addition to rat brain and liver mitochondria induced an increase in lipid peroxidation. These results are discussed in terms of the known physiological differences between mammalian and yeast mitochondria.     

The Control of Iron-Induced Oxidative Damage in Isolated Rat-Liver Mitochondria by Respiration State and Ascorbate

The reaction of iron (II) with H₂O₂ is believed to generate highly reactive species (e.g., OH) capable of initiating biological damage. This study investigates the possibility that the severity of oxidative damage induced by iron in hepatic mitochondria is determined by the level of mitochondrial-H₂O₂ generation, which is believed to be particularly prominent in state-4 respiration.

Iron-induced damage is found to be greater in state-4 than in state-3 respiration. Experiments using uncoupling agents and Ca⁺⁺ to mimic state-3 conditions indicate that this effect reflects differences in the steady-state oxidation-level of the electron carriers of the respiratory chain (and hence the level of H₂O₂ -generation). rather than changes in redox potential or transportation of the metal-ion. Evidence is also presented for a mechanism in which Fe(II) and H₂O₂ react inside the mitochondrial matrix.

Ascorbate (vitamin C) is shown to be pro-oxidant in this system. except when present at very high concentration when it becomes antioxidant in nature.     

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.     

甲基紫精对丹参培养细胞抗氧化防护系统的影响

为了探讨甲基紫精(MV)对丹参(Salvia miltiorrhiza)体内抗氧化防护系统的影响及其生理机制。以MV为诱导剂, 以敌草隆(DCMU)为抑制剂, 考察了MV与DCMU处理后丹参悬浮培养细胞中H₂O₂、丙二醛、还原型谷胱甘肽的含量以及抗氧化防护酶(超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT))活性变化和同工酶的表达差异。结果表明, MV处理显著提高了丹参培养细胞内H₂O₂、丙二醛以及还原型谷胱甘肽含量; MV处理使CAT、POD活性增强, 谱带颜色更亮, 条带增加。DCMU处理显著抑制了MV诱导的H₂O₂、丙二醛、还原型谷胱甘肽含量的增加, 抗氧化酶活性的升高和同工酶的表达。以上结果说明, MV可诱导丹参培养细胞叶绿体产生H₂O₂, H₂O₂激活了丹参培养细胞抗氧化防护系统以维持细胞正常的生理活动。     

Enzymic Pathways Involved in Cell Response to H2O2

An influence of possible interaction of glutathione peroxidase and cyclooxygenase on the clonogenic survival of epithelial cells exposed in vitro to H₂O₂ was investigated. Indomethacin served as the inhibitor of cyclooxygenase, and the use of alkaline (7.5) or acidic (6.5) pH combined with controlled supply of glucose modified glutathione peroxidase activity. Indomethacin affected survival of cells exposed to H₂O₂ in a biphasic manner, enhancing cytotoxicity at lower hydrogen peroxide concentrations, and diminishing it at higher concentrations. The turning point moved gradually to higher concentrations of H₂O₂ corresponding to the augmented decomposition of hydrogen peroxide caused by increased activity of glutathione peroxidase. The data revealed that both enzymic pathways interact in the presence of H₂O₂, resulting in the overall cell survival different from that obtained after inhibition of either.     

Methylmercury and H(2)O(2) provoke lysosomal damage in human astrocytoma D384 cells followed by apoptosis

Methylmercury (MeHg) is a neurotoxic agent acting via diverse mechanisms, including oxidative stress. MeHg also induces astrocytic dysfunction, which can contribute to neuronal damage. The cellular effects of MeHg were investigated in human astrocytoma D384 cells, with special reference to the induction of oxidative-stress-related events. Lysosomal rupture was detected after short MeHg-exposure (1 μM, 1 h) in cells maintaining plasma membrane integrity. Disruption of lysosomes was also observed after hydrogen peroxide (H₂O₂) exposure (100 μM, 1 h), supporting the hypothesis that lysosomal membranes represent a possible target of agents causing oxidative stress. The lysosomal alterations induced by MeHg and H₂O₂ preceded a decrease of the mitochondrial potential. At later time points, both toxic agents caused the appearance of cells with apoptotic morphology, chromatin condensation, and regular DNA fragmentation. However, MeHg and H₂O₂ stimulated divergent pathways, with caspases being activated only by H₂O₂. The caspase inhibitor z-VAD-fmk did not prevent DNA fragmentation induced by H₂O₂, suggesting that the formation of high-molecular-weight DNA fragments was caspase independent with both MeHg and H₂O₂. The data point to the possibility that lysosomal hydrolytic enzymes act as executor factors in D384 cell death induced by oxidative stress.     

NO和H2O2在光/暗调控蚕豆气孔运动中的作用及其相互关系

借助表皮条分析和激光扫描共聚焦显微镜技术,对NO和H2O2在光/暗调控蚕豆(Vicia faba L.)气孔运动中的作用及其相互关系进行了探索.结果显示,光下外源NO供体硝普钠(SNP)和H2O2促进气孔关闭的效应明显大于暗中,暗中NO专一性清除剂2,4-羧基苯-4,4,5,5-四甲基咪唑-1-氧-3-氧化物(cPTIO)、一氧化氮合酶(NOS)抑制剂NG-氮-L-精氨酸-甲酯(L-NAME)和H2O2清除剂抗坏血酸(Vc)、过氧化氢酶(CAT)对气孔开度的效应明显大于光下,而且光下蚕豆保卫细胞NO和H2O2水平比暗中明显降低.上述结果表明,光/暗通过影响保卫细胞NO和H2O2的水平调控气孔运动.研究还发现,光下H2O2既诱导NO水平增加,也诱导气孔关闭,cPTIO和L-NAME有效地逆转H2O2的这些效应;光下SNP既诱导H2O2水平增加,也诱导气孔关闭,SNP的上述效应又被Vc和CAT有效逆转.这些结果表明,NO和H2O2在生成及效应上均存在明显的相互作用.另外,L-NAME显著逆转暗和光下H2O2处理对气孔关闭和NO生成的效应表明,蚕豆保卫细胞中可能存在NOS,暗和光下H2O2处理可能通过提高NOS的活性促进NO水平增加,进而诱导气孔关闭.