Role of thiocyanate, bromide and hypobromous acid in hydrogen peroxide-induced apoptosis

We have previously reported that H₂O₂-induced apoptosis in HL-60 human leukemia cells takes place in the presence of chloride, requires myeloperoxidase (MPO), and occurs through oxidative reactions involving hypochlorous acid and chloramines. We now report that when chloride is replaced by the pseudohalide thiocyanate, there is little or no H₂O₂-induced apoptosis. Furthermore, thiocyanate inhibits H₂O₂-induced apoptosis when chloride is present at physiological concentrations, and this occurs at thiocyanate concentrations that are present in human serum and saliva. In contrast, bromide can substitute for chloride in H₂O₂-induced apoptosis, but results in a lower percent of the cells induced into apoptosis. Hypobromous acid is likely a short-lived intermediate in this H₂O₂/MPO/bromide apoptosis, and reagent hypobromous acid and bromamines induce apoptosis in HL-60 cells. We conclude that the physiologic concentrations of thiocyanate found in human plasma could modulate the cytototoxicity of H₂O₂ and its resulting highly toxic MPO-generated hypochlorous acid by competing with chloride for MPO. Furthermore, the oxidative products of the reaction of thiocyanate with MPO are relatively innocuous for human leukemic cells in culture. In contrast, bromide can support H₂O₂/MPO/halide apoptosis, but is less potent than chloride and it has no effect in the presence of physiological levels of chloride.     

Protective effect of Ecklonia cava enzymatic extracts on hydrogen peroxide-induced cell damage

In this study, Ecklonia cava was enzymatically hydrolyzed to prepare water-soluble extracts, using five carbohydrases (Viscozyme, Celluclast, AMG, Termamyl, and Ultaraflo) and five proteases (Protamex, Kojizyme, Neutase, Flavourzyme, and Alcalase), and the potential antioxidant activity of each was assessed. The Celluclast and Viscozyme extracts of E. cava evidenced good hydrogen peroxide (H₂O₂) scavenging activities (73.25% and 72.92%, respectively) as compared to those of other enzymatic extracts. Therefore, the Celluclast enzymatic extract was selected for use in further experiments, and separated into four different molecular weight fractions (<1, 1–10, 10–30 and >30 kDa). Among these fractions, the >30 kDa fraction manifested the most profound H₂O₂ scavenging activity, with a measured IC₅₀ of 13 μg/ml. The >30 kDa fraction also strongly enhanced cell viability against H₂O₂-induced oxidative damage, and evidenced relatively good lipid peroxidation inhibitory activity in a Chinese hamster lung fibroblast (V79-4) cell line. This fraction also effected a reduction in the proportion of cells undergoing H₂O₂-induced apoptosis, as was demonstrated by a decreased quantity of sub-G₁ hypodiploid cells and decreased apoptotic body formation on the flow cytometry assay. These results clearly indicate that the >30 kDa fraction of E. cava possesses good antioxidant activity against H₂O₂ mediated cell damage in vitro.     

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.     

Effects of tannic acid and its related compounds on food mutagens or hydrogen peroxide-induced DNA strands breaks in human lymphocytes

The effect of tannic acid (TA), gallic acid (GA), propyl gallate (PA) and ellagic acid (EA) on DNA damage in human lymphocytes induced by food mutagens [3-amino-1-methyl-5H-pyrido (4,3-b) indole (Trp-P-2) and 2-amino-1-methyl-6-phenylimadazo (4,5-b) pyridine (PhIP) or H₂O₂ was evaluated by using single-cell electrophoresis (comet assay). The toxicity of these tested compounds (0.1–100 μg/ml) on lymphocytes was not found. These compounds did not cause DNA strand breaks at lower concentrations of 0.1–10 μg/ml. At a concentration of 100 μg/ml, TA and GA exhibited slight DNA damage, whereas PA and EA showed no DNA strand breaks. TA and its related compounds decreased the DNA strand breaks induced by Trp-P-2, PhIP or H₂O₂ at concentrations of 0.1–10 μg/ml. DNA repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycoslase (FPG)] were used to examine the levels of oxidised pyrimidines and purines in human lymphocytes induced by H₂O₂. All the compounds at 10 μg/ml can reduce the level of FPG sensitive sites. However, only EA inhibited the formation of EndoIII sensitive sites. The results indicated that these compounds can enhance lymphocytes resistance towards DNA strand breaks induced by food mutagens or H₂O₂ in vitro.     

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.     

A newly adapted pulsed-field gel electrophoresis technique allows to detect distinct types of DNA damage at low frequencies in human dermal fibroblasts upon exposure to non-toxic H2O2 concentrations

Reactive oxygen species (ROS) comprise several oxygen containing compounds, among them hydrogen peroxide (H₂O₂), which are generated by internal and external sources and play pleiotropic roles in physiological and pathological states. Skin cells as well as cells from other tissues have developed antioxidant defense mechanisms to protect themselves from high concentrations of ROS. Although biological and pathological roles of ROS have previously been elucidated, so far only limited knowledge exists regarding ROS-mediated generation of DNA breaks and base lesions occurring at low frequency in intact skin cells. This study was therefore designed to probe a newly adapted pulsed-field gel electrophoresis technique for the adequate measurement of high molecular weight DNA fragments as well as to investigate the protective role of the antioxidant enzyme catalase against H₂O₂-mediated damage in human dermal fibroblasts. We stably transfected and overexpressed the full-length catalase cDNA in the human dermal fibroblast cell line 1306 in culture and found that these cells are significantly more protected from cytotoxicity, overall DNA strand breaks, and 8-oxodeoxyguanine base lesions resulting from H₂O₂-triggered oxidative stress compared to vector-transfected 1306 cells or secondary dermal fibroblasts. This work has outlined the importance of catalase in the protection from H₂O₂-mediated cytotoxicity and DNA damage which — if unbalanced — even when occurring at low frequency are known to lead to genomic instability, a hallmark in carcinogenesis and premature aging.     

Caspases are reversibly inactivated by hydrogen peroxide

Hydrogen peroxide (H₂O₂) is known to both induce and inhibit apoptosis, however the mechanisms are unclear. We found that H₂O₂ inhibited the activity of recombinant caspase-3 and caspase-8, half-inhibition occurring at about 17 μM H₂O₂. This inhibition was both prevented and reversed by dithiothreitol while glutathione had little protective effect. 100–200 μM H₂O₂ added to macrophages after induction of caspase activation by nitric oxide or serum withdrawal substantially inhibited caspase activity. Activation of H₂O₂-producing NADPH oxidase in macrophages also caused catalase-sensitive inactivation of cellular caspases. The data suggest that the activity of caspases in cells can be directly but reversibly inhibited by H₂O₂.     

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.     

Effect of oxygen transfer rate on β-carotene production from synthetic medium by Blakeslea trispora in shake flask culture

The effect of oxygen transfer rate (OTR) on β-carotene production by Blakelsea trispora in shake flask culture was investigated. The results indicated that the concentration of β-carotene (704.1 mg/l) was the highest in culture grown at maximum OTR of 20.5 mmol/(l h). In this case, the percentage of zygospores was over 50.0% of the biomass dry weight. On the other hand, OTR level higher than 20.5 mmol/(l h) was found to be detrimental to cell growth and pigment formation. To elucidate the effect of oxidative stress on β-carotene synthesis, the accumulation of hydrogen peroxide during fermentation under different OTRs was determined. A linear response of β-carotene synthesis to the level of H₂O₂ was observed, indicating that β-carotene synthesis is stimulated by H₂O₂. However, there was an optimal concentration of H₂O₂ (2400 μM) in enhancing β-carotene synthesis. At a higher concentration of H₂O₂, β-carotene decreased significantly due to its toxicity.     

Nitric oxide (as sodium nitroprusside) supplementation ameliorates Cd toxicity in hydroponically grown wheat roots

Cadmium (Cd) is a non-redox toxic heavy metal present in the environment and induces oxidative stress in plants. We investigated whether exogenous nitric oxide (NO) supplementation as sodium nitroprusside (SNP) has any ameliorating action against Cd-induced oxidative damage in plant roots and thus protective role against Cd toxicity. Cd treatment (50 or 250 μM) alone or in combination with 200 μM SNP was given to hydroponically grown wheat roots for a short time period of 24 h and then these were shifted to distilled water to observe changes in levels of oxidative markers (lipid peroxidation, H₂O₂ content and electrolyte leakage). Supplementation of Cd with SNP significantly reduced the Cd-induced lipid peroxidation, H₂O₂ content and electrolyte leakage in wheat roots. It indicated a reactive oxygen species (ROS) scavenging activity of NO. However, even upon removal of Cd-treatment solution, the levels of oxidative markers increased during 24 h recovery stage and later at 48 h these decreased. Cd treatment resulted in an upregulation of activities of antioxidant enzymes—superoxide dismutase (SOD, 1.15.1.1), guaiacol peroxidase (GPX, 1.11.1.7), catalase (CAT, 1.11.1.6), and glutathione reductase (GR, 1.6.4.2). SNP supply resulted in a reduction in Cd-induced increased activities of scavenging enzymes. The protective role of exogenous NO in decreasing Cd-induced oxidative damage was also evident from the histochemical localization of lipid peroxidation, plasma membrane integrity and superoxides. The study concludes that an exogenous supply of NO protects wheat roots from Cd-induced toxicity.     

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     

A thermodynamic and crystallographic study of complexes of the highly preorganized ligand 8-hydroxyquinoline-2-carboxylic acid

The metal ion complexing properties of the ligand HQC (8-hydroxyquinoline-2-carboxylic acid) are reported. The structures of [Zn(HQCH)₂] · 3H₂O (1) and [Cd(HQCH)₂] · 3H₂O (2) were determined (HQCH = HQC with phenol protonated). Both 1 and 2 are triclinic, space group , with Z = 2. For 1 a = 7.152(3), b = 9.227(4), c = 15.629(7) Å,  = 103.978(7)°, β = 94.896(7)°, γ = 108.033(8)°, R = 0.0499. For 2 a = 7.0897(5), b = 9.1674(7), c = 16.0672(11) Å,  = 105.0240(10)°, β = 93.9910(10)°, γ = 107.1270(10)°, R = 0.0330. In 1 the Zn has a distorted octahedral coordination geometry, with Zn–N of 2.00 and 2.15 Å, and Zn–O to the protonated phenolic oxygens of 2.431 and 2.220 Å. The structure of 2 is similar, with Cd–N bonds of 2.220 and 2.228 Å, with Cd–O bonds to the protonated phenolate oxygens of 2.334 and 2.463 Å. The structures of 1 and 2, and isomorphous Ni(II) and Co(II) HQC complexes reported in the literature, show very interesting short (<2.5 Å) O–O distances in H-bonds involving the protons on the coordinated phenolates and lattice water molecules. These are discussed in relation to the possible role of short low-energy H-bonds in alcohol dehydrogenase in mediating the transfer of the hydroxyl proton of the alcohol to an adjacent serine oxygen.

The formation constants for HQC are determined by UV–Visible spectroscopy at 25 °C in 0.1 M NaClO₄ with Mg(II), Ca(II), Sr(II), Ba(II), La(III), Gd(III), Zn(II), Cd(II), Ni(II), Cu(II), and Pb(II). These show greatest stabilization with metal ions with an ionic radius above 1.0 Å. This is as would be expected from the fact that HQC forms two five-membered chelate rings on complex-formation, which favors larger metal ions. The ligand design concept of using rigid aromatic backbones in ligands to achieve high levels of preorganization, and hence the high log K values (for a tridentate ligand) and strong metal ion selectivities observed for HQC, is discussed.     

Hydrogen Peroxide Modification of Human Oxyhemoglobin

The effect of H₂O₂ on the primary structure of OxyHb was studied. Upon treatment of Oxy Hb with H₂O₂ ([Heme]/[H₂O₂] =I), tryptophan and methionine residues of the /-chain were modified. Treatment of ApoHb with H₂O₂ resulted in the modification of histidine and methionine residues in both globin chains. Tryptophan residues were unaffected. Modification of methionine residues in both the β-chain of OxyHb and ApoHb probably results from the direct oxidation of mcthionine by H₂O₂. The modification of histidine residues in ApoHb may be mediated by a metal-catalyzed oxidation system comprised of H₂O₂ and histidine-bound iron. The H₂O₂-mediated modification of tryptophan in the OxyHb β-chain. however, requires the heme moiety.     

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.     

Fabrication of a miniature CMOS-based optical biosensor

This work presents a novel, miniature optical biosensor by immobilizing horseradish peroxidase (HRP) or the HRP/glucose oxidase (GOx) coupled enzyme pair on a CMOS photosensing chip with a detection area of 0.5 mm × 0.5 mm. A highly transparent TEOS/PDMS Ormosil is used to encapsulate and immobilize enzymes on the surface of the photosensor. Interestingly, HRP-catalyzed luminol luminescence can be detected in real time on optical H₂O₂ and glucose biosensors. The minimum reaction volume of the developed optical biosensors is 10 μL. Both optical H₂O₂ and glucose biosensors have an optimal operation temperature and pH of 20–25 °C and pH 8.4, respectively. The linear dynamic range of optical H₂O₂ and glucose biosensors is 0.05–20 mM H₂O₂ and 0.5–20 mM glucose, respectively. The miniature optical glucose biosensor also exhibits good reproducibility with a relative standard deviation of 4.3%. Additionally, ascorbic acid and uric acid, two major interfering substances in the serum during electrochemical analysis, cause only slight interference with the fabricated optical glucose biosensor. In conclusion, the CMOS-photodiode-based optical biosensors proposed herein have many advantages, such as a short detection time, a small sample volume requirement, high reproducibility and wide dynamic range.     

Two new multi-cobalt-containing heteropolyoxotungstates

Two new multi-cobalt-containing polyoxotungstates K₄Na₆Co₂(H₂O)₁₂{Co(H₂O)₄[Co₂(H₂O)₁₀Co₄(H₂O)₂(B--SiW₉O₃₄)₂]₂} · 40H₂O (1) and K₁₀Na₂[Co₄(H₂O)₂(GeW₉O₃₄)₂] · 20H₂O (2) have been obtained by the routine synthetic reactions in aqueous solution. The polyoxoanion framework of 1 consists of two sandwich-type polyoxoanions [Co₄(H₂O)₂(B--SiW₉O₃₄)₂]¹²⁻ connected together by a [CoO₂(H₂O)₄] cluster to constitute the sandwich dimer, and then, four isolated Co(H₂O)₅ cations coordinate to the dimer through four μ₂-O atoms. The polyoxoanion 2 is isomorphic to the sandwich-type polyoxoanion [Co₄(H₂O)₂(B--SiW₉O₃₄)₂]¹²⁻ in 1. The magnetic property of compound 1 has been studied by measuring its magnetic susceptibility in the temperature range 2.0–300.0 K, indicating the existence of intramolecular ferromagnetic Co–Co interactions, and, the electrochemical properties of 1 and 2 are detected in the pH 4 buffer solution.     

Electrochemical determination of uric acid at ordered mesoporous carbon functionalized with ferrocenecarboxylic acid-modified electrode

Ordered mesoporous carbon (OMC) functionalized with ferrocenecarboxylic acid (Fc) was used to modify the glassy carbon (GC) electrode. The characterization of OMC–Fc shows that, after anchoring ferrocene on the mesoporous, ordered mesostructure of the material (OMC–Fc) remains intact and Fc is electrochemically accessible. The obtained OMC–Fc-modified electrode was used to investigate the electrochemical behavior of uric acid (UA). UA oxidation is catalyzed by this electrode in aqueous buffer solution (pH 7.3) with a decrease of 200 mV in overpotential compared to GC electrode. The detection and determination of UA in the presence of ascorbic acid (AA), the main interferent, were achieved. The voltammetric signals due to UA and AA were well separated with a potential difference of 308 mV, a separation that can allow the simultaneous determination of UA and AA. With amperometric method, at a constant potential of 375 mV, the catalytic current of UA versus its concentration shows a good linearity in the range 60–390 μM (R = 0.998) with a detection limit of 1.8 μM (S/N = 3). These results are not influenced by the presence of AA in the sample solution. With good stability and reproducibility, the present OMC–Fc-modified electrode was applied in the determination of UA content in urine sample and satisfactory results were obtained.     

Application of confocal laser scanning microscopy to detect oxidative stress in human colon cells

Introduction Excess of intracellular reactive oxygen species in relation to antioxidative systems results in an oxidative environment which may modulate gene expression or damage cellular molecules. These events are expected to greatly contribute to processes of carcinogenesis. Only few studies are available on the oxidative/reductive conditions in the colon, an important tumour target tissue. It was the objective of this work to further develop methods to assess intracellular oxidative stress within human colon cells as a tool to study such associations in nutritional toxicology.

Methods We have measured H₂O₂-induced oxidative stress in different colon cell lines, in freshly isolated human colon crypts, and, for comparative purposes, in NIH3T3 mouse embryo fibroblasts. Detection was performed by loading the cells with the fluorigenic peroxide-sensitive dye 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (diacetoxymethyl ester), followed by in vitro treatment with H₂O₂ and fluorescence detection with confocal laser scanning microscopy (CLSM). Using the microgel electrophoresis (“Comet”) Assay, we also examined HT29 stem and clone 19A cells and freshly isolated primary colon cells for their relative sensitivity toward H₂O₂-induced DNA damage and for steady-state levels of endogenous oxidative DNA damage.

Results A dose-response relationship was found for the H₂O₂-induced dye decomposition in NIH3T3 cells (7.8-125 μM H₂O₂) whereas no effect occurred in the human colon tumour cell lines HT29 stem and HT29 clone 19A (62-1000 μM H₂O₂). Fluorescence was significantly increased at 62 μM H₂O₂ in the human colon adenocarcinoma cell line Caco-2. In isolated human colon crypts, the lower crypt cells (targets of colon cancer) were more sensitive towards H₂O₂ than the more differentiated upper crypt cells. In contrast to the CLSM results, oxidative DNA damage was detected in both cell lines using the Comet Assay. Endogenous oxidative DNA damage was highest in HT29 clone 19A, followed by the primary colon cells and HT29 stem cells.

Conclusions Oxidative stress in colon cells leads to damage of macromolecules which is sensitively detected in the Comet Assay. The lacking response of the CLSM-approach in colon tumour cells is probably due to intrinsic modes of protective activities of these cells. In general, however, the CLSM method is a sensitive technique to detect very low concentrations of H₂O₂-induced oxidative stress in NIH3T3 cells. Moreover, by using colon crypts it provides the unique possibility of assessing cell specific levels of oxidative stress in explanted human tissues. Our results demonstrate that the actual target cells of colon cancer induction are indeed susceptible to the oxidative activity of H₂O₂.     

Hydrogen peroxide-induced apoptosis in HL-60 cells requires caspase-3 activation

Apoptosis has been associated with oxidative stress in biological systems. Caspases have been considered to play a pivotal role in the execution phase of apoptosis. However, which caspases function as executioners in reactive oxygen species (ROS)-induced apoptosis is not known. The present study was performed to identify the major caspases acting in ROS-induced apoptosis. Treatment of HL-60 cells with 50 μM hydrogen peroxide (H₂O₂) for 4 h induced the morphological changes such as condensed and/or fragmented nuclei, increase in caspase-3 subfamily protease activities, reduction of the procaspase-3 and a DNA fragmentation. To determine the role of caspases in H₂O₂-induced apoptosis, caspase inhibitors, acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone(Ac-YVAD-cmk), acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) and acetyl-Val-Glu-lle-Aspaldehyde (Ac-VEID-CHO), selective for caspase-1 subfamily, caspase-3 subfamily and caspase-6, respectively, were loaded into the cells using an osmotic lysis of pinosomes method. Of these caspase inhibitors, only Ac-DEVD-CHO completely blocked morphological changes, caspase-3 subfamily protease activation and DNA ladder formation in H₂O₂-treated HL-60 cells. This inhibitory effect was dose-dependent. These results suggest that caspase-3, but not caspase-1 is required for commitment to ROS-triggered 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。