Glutathione-dependent generation of reactive oxygen species by the peroxidase-catalyzed redox cycling of flavonoids.

Catalytic concentrations of apigenin (a flavone containing a phenol B ring) and naringin or naringenin (flavanones containing a phenol B ring) caused extensive GSH oxidation at a physiological pH in the presence of peroxidase. Only catalytic H2O2 concentrations were required, indicating a redox cycling mechanism that generated H2O2 was involved. Extensive oxygen uptake ensued, the extent of which was proportional to the extent of GSH oxidation to GSSG and was markedly increased by superoxide dismutase. These results suggest that prooxidant phenoxyl radicals formed by these flavonoids co-oxidized GSH to form thiyl radicals which activated oxygen. GSH also prevented the peroxidase-catalyzed oxidative destruction of these flavonoids which suggests that phenoxyl radicals initiated the oxidative destruction. This is the first time that a group of flavonoids have been identified as prooxidants independent of autoxidation reactions catalyzed by the transition metal ions Fe3+, Fe2+, Mn2+, and Cu2+.     

The role of iron in reactive oxygen species generation from diesel exhaust particles.

Diesel exhaust particles (DEP) are known to produce reactive oxygen species (ROS), which induce oxidative stress and inflammation in the lung and respiratory tract. DEP are composed of polycyclic aromatic hydrocarbons (PAH) and their derivatives, redox active semi-quinones, and trace amounts of heavy metals. ROS production was measured by thiobarbituric acid-reactive substances of deoxyribose (TBARS) formation from DEP samples obtained from Korea (DEP-KO), and the Standard Reference Material (SRM) 2975 to explore the role of transition metals. Both DEP-KO and SRM2975 had similar amounts of transition metals, whereas DEP-KO contained more iron, but less copper and zinc, than SRM2975. The water-soluble fraction from SRM2975, but not that from DEP-KO, had a broad absorption in the visible region, but not from DEP-KO, obscuring an accurate absorption measurement of TBARS. Fluorescence measurements of TBARS generation in a water-soluble extract showed that SRM2975 produces more TBARS, but the addition of hydrogen peroxide (H2O2) generated more TBARS in DEP-KO than in SRM2975, consistent with the higher amounts of iron in DEP-KO. The incubation of DEP with iron chelators inhibited the production of TBARS. Finally, a novel use of the fluorogenic spin trap probe, proxyl fluorescamine, enabled the detection of the ROS production from both DEP-KO and SRM2975. Our findings suggested that careful consideration is needed to measure TBARS production in DEP, and that iron in DEP seems to be more important than other transition metals in H2O2-induced ROS generation.     

Generation of photoemissive species by mitomycin C redox cycling in rat liver microsomes

Generation of reactive oxygen species during redox cycling is thought to be involved in the chemotherapeutic action of quinone anticancer drugs. A clinically used agent which contains a quinone moiety is mitomycin C (Mit C). With isolated rat liver microsomes we detected photoemissive species during Mit C-induced redox cycling. After addition of reduced glutathione (GSH) a large increase in Mit C-induced chemiluminescence was observed. The increase of photoemission in deuterium oxide as well as greater than 90% of intensity at wavelengths greater than 610 nm suggest that singlet oxygen is a photoemissive species generated by this system. Glutathione disulfide (GSSG) accumulates during the reaction. We propose that superoxide anion radicals formed during redox cycling of Mit C react with GSH. Generation of glutathionyl radicals followed by oxygen addition then leads to the formation of photoemissive species and GSSG.     

Intracellular generation of reactive oxygen species by mitochondria

Mitochondria have bioenergetic properties that strongly suggest their involvement in the cellular formation of reactive oxygen species (ROS). Apparent confirmation of this process has come from work with isolated mitochondria, which have been shown to produce H(2)O(2) from dismutating superoxide radicals. Two different sites were reported to shuttle single electrons to oxygen out of the normal respiratory sequence. However, the mechanisms for ROS formation at these two sites are controversial. Arguments against mitochondrial ROS formation in the living cell are based on the fact that bioenergetic alterations may result from the mechanical removal of mitochondria from their natural environment. Furthermore, the invasive detection methods that are generally used may be inappropriate because of the possible interaction of the detection system with mitochondrial constituents. The use of non-invasive detection methods has proved that ROS formation does not occur unless changes in the physical state of the membrane are established. The aim of this commentary is to discuss critically the arguments in favor of mitochondria as the main intracellular source of ROS. The pros and cons of working with isolated mitochondria, as well as the detection methodology are carefully analyzed to judge whether or not the above assumption is correct. The conclusion that mitochondria are the main ROS generators in the cell contradicts the fact that ROS release was not observed. However, if electron flow from ubiquinol to the bc(1) complex is hindered due to changes in lipid fluidity, single electrons may transfer to dioxygen and produce H(2)O(2) via superoxide radicals.     

Inherent redox properties of diesel exhaust particles: catalysis of the generation of reactive oxygen species by biological reductants.

The toxicity of diesel exhaust particles (DEP) can be due to the particle itself, extractable components, or both. Many studies focus on the biological properties of DEP-extractable components although it is possible that chemical properties inherent to the DEP itself can lead to toxicity. Thus, an examination of the chemistry inherent to DEP was carried out. Herein, we report that DEP are capable of catalyzing the consumption of O2 (monitored using a Clarke electrode) by ascorbate and thiols leading to the generation of reactive oxygen species. Consistent with the idea that DEP are capable of catalyzing the generation of reactive oxygen species, they were also found to catalyze DNA strand breakage via an O2- and reductant-dependent process. Significantly, extraction of DEP with either organic solvent (methylene chloride) or acid (aqueous HCl) did little to abrogate this chemistry. Finally, using electron paramagnetic spectrometry (EPR), DEP were found to have paramagnetic properties. The paramagnetic character of DEP may be important to their ability to catalyze the formation of reactive oxygen species and at least partially responsible for their toxicity. These findings indicate that studies that primarily consider or examine particle extracts as the toxic components of DEP may be insufficient in describing the toxicity associated with DEP exposure.     

Mitochondrial generation of reactive oxygen species and its role in aerobic life

Mitochondria are the major site for the generation of ATP at the expense of molecular oxygen. Significant fractions (approximately 2%) of oxygen are converted to the superoxide radical and its reactive metabolites (ROS) in and around mitochondria. Although ROS have been known to impair a wide variety of biological molecules including lipids, proteins and DNA, thereby causing various diseases, they also play critical roles in the maintenance of aerobic life. Because mitochondria are the major site of free radical generation, they are highly enriched with antioxidants including GSH and enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase, on both sides of their membranes to minimize oxidative stress in and around this organelle. The present work reviews the sites and mechanism of ROS generation by mitochondria, mitochondrial localization of Mn-SOD and Cu,Zn-SOD which has been postulated for a long time to be a cytosolic enzyme. The present work also describes that a cross-talk of molecular oxygen, nitric oxide (NO) and superoxide radicals regulates the circulation, energy metabolism, apoptosis, and functions as a major defense system against pathogens. Pathophysiological significance of ROS generation by mitochondria in the etiology of aging, cancer and degenerative neuronal diseases is also described.     

Pathophysiological and pharmacological implications of mitochondria-targeted reactive oxygen species generation in astrocytes

Astrocytes, in addition to passively supporting neurons, have recently been shown to be actively involved in synaptic transmission and neurovascular coupling in the central nervous system (CNS). This review summarizes briefly our previous observations using fluorescent probes coupled with laser scanning digital imaging microscopy to visualize spatio-temporal alteration of mitochondrial reactive oxygen species (mROS) generation in intact astrocytes. mROS formation is enhanced by exogenous oxidants exposure, Ca(2+) stress and endogenous pathological defect of mitochondrial respiratory complexes. In addition, mROS formation can be specifically stimulated by visible light or visible laser irradiation and can be augmented further by photodynamic coupling with photosensitizers, particularly with mitochondria-targeted photosensitizers. "Severe" oxidative insult often results in massive and homogeneous augmentation of mROS formation which causes cessation of mitochondrial movement, pathological fission and irreversible swelling of mitochondria and eventually apoptosis or necrosis of cells. Mitochondria-targeted antioxidants and protectors such as MitoQ, melatonin and nanoparticle C(60) effectively prevent "severe" mROS generation. Intriguingly, "minor" oxidative insults enhance heterogeneity of mROS and mitochondrial dynamics. "Minor" mROS formation-induced fission and fusion of mitochondria relocates mitochondrial network to form a mitochondria free gap, i.e., "firewall", which may play a crucial role in mROS-mediated protective "preconditioning" by preventing propagation of mROS during oxidative insults. These mROS-targeted strategies for either enhancement or prevention of mitochondrial oxidative stress in astrocytes may provide new insights for future development of therapeutic interventions in the treatment of cancer such as astrocytomas and gliomas and astrocyte-associated neurodegeneration, mitochondrial diseases and aging.     

Oxidation of ethylene glycol to formaldehyde by rat liver microsomes. Role of cytochrome P-450 and reactive oxygen species.

Rat liver microsomes oxidized ethylene glycol to formaldehyde in a NADPH-dependent, carbon monoxide-sensitive manner. Formaldehyde production was inhibited by substrates and ligands for cytochrome P-450 such as aniline, p-nitrophenol, pyrazole, and 4-methylpyrazole, and inhibitors such as tryptamine, cimetidine, and miconazole. The apparent Km for ethylene glycol was about 25 mM and the apparent Vmax was about 6 nmol/min/mg protein. Microsomes isolated from rats treated with pyrazole or 4-methylpyrazole to induce cytochrome P-450IIE1 oxidized ethylene glycol at rates which were about twice those found with control microsomes or microsomes isolated from rats treated with phenobarbital or 3-methylcholanthrene, although significant rates were found with all microsomal preparations. Antibody raised against the pyrazole-induced P-450IIE1 inhibited formaldehyde production from ethylene glycol in microsomes from pyrazole-treated rats. H2O2 itself did not oxidize ethylene glycol to formaldehyde; however, the microsomal reaction was inhibited by catalase or glutathione plus glutathione peroxidase and was stimulated by added H2O2 in the presence of NADPH. Nonheme iron also appeared to be required for ethylene glycol oxidation in view of the inhibition of formaldehyde production by desferrioxamine, EDTA, and DTPA. Microsomal oxidation of ethylene glycol was not sensitive to superoxide dismutase, hydroxyl radical scavengers, or Trolox, suggesting that the oxidant derived from H2O2 and iron and responsible for the production of formaldehyde from ethylene glycol was not superoxide, hydroxyl radical, or lipid hydroperoxide. These results suggest that ethylene glycol is oxidized to formaldehyde by an oxidant derived from H2O2 and nonheme iron, and that cytochrome P-450 may function to generate the H2O2 and to catalyze reduction of the nonheme iron.     

Berberine-induced apoptosis in human prostate cancer cells is initiated by reactive oxygen species generation

Phytochemicals show promise as potential chemopreventive or chemotherapeutic agents against various cancers. Here we report the chemotherapeutic effects of berberine, a phytochemical, on human prostate cancer cells. The treatment of human prostate cancer cells (PC-3) with berberine induced dose-dependent apoptosis but this effect of berberine was not seen in non-neoplastic human prostate epithelial cells (PWR-1E). Berberine-induced apoptosis was associated with the disruption of the mitochondrial membrane potential, release of apoptogenic molecules (cytochrome c and Smac/DIABLO) from mitochondria and cleavage of caspase-9,-3 and PARP proteins. This effect of berberine on prostate cancer cells was initiated by the generation of reactive oxygen species (ROS) irrespective of their androgen responsiveness, and the generation of ROS was through the increased induction of xanthine oxidase. Treatment of cells with allopurinol, an inhibitor of xanthine oxidase, inhibited berberine-induced oxidative stress in cancer cells. Berberine-induced apoptosis was blocked in the presence of antioxidant, N-acetylcysteine, through the prevention of disruption of mitochondrial membrane potential and subsequently release of cytochrome c and Smac/DIABLO. In conclusion, the present study reveals that the berberine-mediated cell death of human prostate cancer cells is regulated by reactive oxygen species, and therefore suggests that berberine may be considered for further studies as a promising therapeutic candidate for prostate cancer.     

The effect of adherence on the generation of reactive oxygen species by human neutrophilic granulocytes

Human neutrophilic granulocytes (PMN) suspended in protein containing salt solution or adherent on protein coated nylon fibers were tested for the production of H2O2 and O2- in response to various PMN stimulants. Upon stimulation with the chemotactic factors formyl-methionyl-leucyl-phenylalanine, C5a and platelet activating factor, the non-chemotactic ionophore A23187, and the chemotaxis inhibitors tumor necrosis factor (TNF alpha) and lymphotoxin (TNF beta) adherent PMN produced considerably more reactive oxygen metabolites than suspended cells. The relative amounts of the two metabolites varied with the stimulus and its concentration, TNF alpha and TNF beta favoring H2O2 production, C5a eliciting more O2- than H2O2 and the other active stimulants being in between. Leukotriene B4 and a novel monocyte-derived chemotaxin were inactive in releasing either oxygen derivative from adherent or suspended PMN. The data indicate that attachment of PMN to endothelial cells or to connective tissue substances can strongly enhance its ability to respond to a given stimulus with the production of reactive oxygen metabolites. The findings may in part explain the "priming" phenomenon since many PMN-priming mediators increase the cells' adherence.     

Bisphenol A induces reactive oxygen species generation in the liver of male rats

Bisphenol A, an environmental contaminant, widely used as a monomer in polycarbonate plastics, has been shown to cause abnormalities in liver of rats and mice. The nature and mechanism of action of bisphenol A on liver is not clear. The aim of the present study was to investigate if bisphenol A induces oxidative stress in the liver of rats and if co-administration of vitamin C, an antioxidant, can prevent oxidative stress. Bisphenol A (0.2, 2.0 and 20 micro g/kg body weight per day) and bisphenol A+vitamin C (0.2, 2.0, 20 micro g+40 mg/kg body weight per day) was orally administered to rats for 30 days. After 24 h of the last treatment, rats were killed using overdose of anesthetic ether. Body weights of the animals and the weights of liver showed no significant changes. The activities of antioxidant enzymes, superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase were decreased in mitochondrial and microsome-rich fractions of liver. The levels of hydrogen peroxide and lipid peroxidation increased in the treated rats when compared with the corresponding group of control animals. Activity of alanine transaminase, a marker enzyme of hepatic injury remained unchanged in the treated rats as compared with the corresponding control rats. Co-administration of bisphenol A and vitamin C showed no changes in the activities of superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase and in the levels of hydrogen peroxide and lipid peroxidation as compared with the corresponding control groups. The results indicated that bisphenol A induces oxidative stress in the liver of rats by decreasing the antioxidant enzymes. Co-administration of vitamin C reversed the effects of bisphenol A-induced oxidative stress in the liver of rats.     

Requirement of reactive oxygen species generation in apoptosis of leukemia cells induced by 2-methoxyestradiol

Aim： To investigate the effects of 2-methoxyestradiol （2-ME） on 2 myeloid leukemia cell lines HL-60 and U937, and to explore its mechanisms. Methods： Human myeloid leukemia cells HL-60 and U937 were used. Measurement ofmitochondrial membrane potential （Dym） was performed using 5,5′＇,6,6′-Tetrachloro-1, 1′,3,3′- tetraethylbenzimidazolylcarbocyanine iodide （ JC- 1）. Apoptosis and cellular nitric oxide （NO） were detected by flow cytometry using Annexin V and NO sensor dye. Superoxide anion was measured with a fluorescent plate reader by dihydroethidium （DHE）. Cytotoxicity was analyzed by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl- tetrazolium assay. Results： 2-ME resulted in viability decrease in a dose-dependent manner. 2-ME treatment also generated reactive oxygen species （ROS）, including NO and superoxide anions, which resulted in mitochondria damage. 2-ME-induced apoptosis was correlated with an increase in ROS. The quenching of ROS with N-acetyl-L-cysteine protected leukemia cells from 2-ME cytotoxicity and prevented apoptosis induction by 2-ME. Furthermore, the addition of manumycin, a farnesyltransferase inhibitor, significantly enhanced apoptosis induced by 2-ME. Conclusion： Cellular ROS generation plays an important role in the cytotoxic effect of 2-ME. It is possible to use ROS generation agents, such as manumycin, to enhance the antileukemic effect. The combination strategy needs further in vivo justification and may have potential clinical application.     

Effects of toluene and its metabolites on cerebral reactive oxygen species generation

The effects of toluene on lipid peroxidation and rates of reactive oxygen species (ROS) formation have been studied in isolated systems and in vivo. The induction of reactive oxygen species was assayed using the probe 2',7'-dichlorofluorescin diacetate (DCFH-DA). Toluene exposure (1 g/kg, 1 hr, i.p.) did not stimulate cortical lipid peroxidation as evaluated by measurement of conjugated dienes. Exposure to toluene, however, both in vivo and in vitro, caused a significant elevation of ROS formation within cortical crude synaptosomal fractions (P2) and microsomal fractions (P3). The ROS-inducing properties of toluene were blocked in vivo in the presence of a mixed-function oxidase inhibitor, metyrapone. This suggested that a metabolite of toluene may catalyze reactive oxygen formation. Both benzyl alcohol and benzoic acid, in vitro, were found to have free radical quenching properties, while benzaldehyde exhibited significant induction of ROS generation. It appears that benzaldehyde is the metabolite responsible for the effect of toluene in accelerating reactive oxygen production within the nervous system. Benzaldehyde may also contribute to the overall neurotoxicity of toluene.     

Prooxidant action of rosmarinic acid: transition metal-dependent generation of reactive oxygen species.

Rosmarinic acid and its constituent caffeic acid produced reactive oxygen species in the presence of transition metals. Complex of rosmarinic acid or caffeic acid with iron inactivated aconitase the most sensitive enzyme to oxidative stress. The inactivation of aconitase was iron-dependent, and prevented by TEMPOL, a scavenger of reactive oxygen species, suggesting that the rosmarinic acid/iron-mediated generation of superoxide anion is responsible for the inactivation of aconitase. Direct spectrophotometric determination of hydrogen peroxide and superoxide anion confirmed the rosmarinic acid/iron-dependent production of reactive oxygen species. Treatment of DNA from plasmid pBR322 and calf thymus with rosmarinic acid plus copper caused strand scission and formed 8-hydroxy-2'-deoxyguanosine in DNA. Rosmarinic acid and caffeic acid showed a potent activity that reduces transition metals. These results suggest that transition metals reduced by rosmarinic acid can form superoxide radical by one electron reduction of oxygen molecule: superoxide radical in turn converts to hydrogen peroxide and hydroxyl radical causing the formation of DNA base adduct. Cytotoxicity of rosmarinic acid may be related to the prooxidant action resulting from metal-reducing activity.     

Discriminating redox cycling and arylation pathways of reactive chemical toxicity in trout hepatocytes.

The toxicity of four quinones, 2,3-dimethoxy-1,4-naphthoquinone (DMONQ), 2-methyl-1,4-naphthoquinone (MNQ), 1,4-naphthoquinone (NQ), and 1,4-benzoquinone (BQ), which redox cycle or arlyate in mammalian cells, was determined in isolated trout (Oncorhynchus mykiss) hepatocytes. More than 70% of cells died in 3 h when exposed to BQ or NQ; 50% died in 7 h when exposed to MNQ, with no mortality compared to controls after 7 h DMONQ exposure. A suite of biochemical parameters was assessed for ability to discriminate these reactivity pathways in fish. Rapid depletion of glutathione (GSH) with appearance of glutathione disulfide (GSSG) and increased dichlorofluoroscein fluorescence were used as indicators of redox cycling, noted with DMONQ, MNQ, and NQ. Depletion of GSH with no GSSG accumulation, and loss of free protein thiol (PrSH) groups (nonreducible) indicated direct arylation by BQ. All toxicants rapidly oxidized NADH, with changes in NADPH noted later (BQ, NQ, MNQ) or not at all (DMONQ). Biochemical measures including cellular energy status, cytotoxicity, and measures of reactive oxygen species, along with the key parameters of GSH and PrSH redox status, allowed differentiation of responses associated with lethality. Chemicals that arylate were more potent than redox cyclers. Toxic pathway discrimination is needed to group chemicals for potency predictions and identification of structural parameters associated with distinct types of reactive toxicity, a necessary step for development of mechanistically based quantitative structure-activity relationships (QSARs) to predict chemical toxic potential. The commonality of reactivity mechanisms between rodents and fish was also demonstrated, a step essential for species extrapolations.     

Mechanism of DNA damage and apoptosis induced by anticancer drugs through generation of reactive oxygen species

A number of anticancer drugs exert their effect by causing DNA damage and subsequent apoptosis induction. Reactive oxygen species (ROS), such as hydrogen peroxide (H(2)O(2)) and super oxide anion (O(2)(-)), participate in apoptosis and DNA damage induced by some anticancer drugs, however, the precise mechanism of apoptosis via ROS formation remains to be clarified. I investigated the mechanism of apoptosis and DNA damage induced by anticancer drugs, especially topoisomerase inhibitors, using human cultured cells. TAS-103, a topoisomerase inhibitor, induces apoptosis through DNA cleavage and subsequent H(2)O(2) generation mediated by poly (ADP-ribose) polymerase (PARP) and NAD(P)H oxidase activation. Doxorubicin (DOX), an anthracycline antibiotic and topoisomerase inhibitor, induces apoptosis through direct oxidative DNA damage leading to indirect H(2)O(2) generation mediated by PARP and NAD(P)H oxidase activation. DOX caused site-specific oxidative DNA damage in the presence of copper(II), which may contribute to apoptosis. These findings suggest that ROS formation plays important roles in apoptosis induced by anticancer drugs. Furthermore, these studies may provide an insight into the development of new effective chemotherapeutic drugs.     

Effect of adrenergic agents on NADPH oxydase evoked production of reactive oxygen species by macrophages

The effects of adrenergic agonists (epinephrine, norepinephrine, isoprenaline, salbutamol, phenylephrine, clonidine) and antagonists (propranolol, sotalol, oxprenolol, methoprolol, atenolol) on the production of reactive oxygen (RO) by macrophages (MFs) were studied in vitro using chemiluminescence techniques. It was established that beta-adrenomimetics suppressed, whereas alpha-adrenomimetics enhanced or did not change the RO, production by MFs. Possible mechanisms and the biological significance of the adrenergic modulation NADPH-dependent RO, generation are discussed.     

Small-molecule anthracene-induced cytotoxicity and induction of apoptosis through generation of reactive oxygen species

A series of anthracene derivatives have been synthesized, and their potential individual cytotoxicity was evaluated using Jurkat T cells and peripheral blood mononuclear cells (PBMCs) in vitro. These compounds, except for 2l, showed less cytotoxicity in PBMCs than mitoxantrone. We also analyzed the antiproliferative activity of these derivatives using the annexin V/propidium iodide assay. These synthetic compounds induced apoptosis, thus leading to antitumor effects. Compounds 2b, 2e, 2f, 2g, 2h, 2i, 2j, and mitoxantrone produced dose-dependent cytotoxicity, while the antiproliferative activity of the anthracene pharmacophore was retained in Jurkat T cells base on the detection of DNA degradation and membrane unpacking. These clearly indicate a correlation between cytotoxicity and antitumor activity. Unlike mitoxantrone, cytotoxic properties were observed, as documented by the reactivity of these novel compounds against Jurkat T cells and PBMCs as normal cells, respectively. Various concentrations of 2b, 2e, 2f, 2g, 2h, 2i, and 2j preparations also inhibited Jurkat T cell proliferation and induced apoptosis of Jurkat T cells, potentially confirmed through the detection of DNA degradation and membrane unpacking. In the present report we also investigated the antiinflammatory activity against phorbol-12-myristate-13-acetate induced superoxide anion production, a marker for an inflammatory mediator produced by neutrophils, with IC(50) (microM) values of 2b, 2h, 2l, and 2o of 4.28+/-0.89, 3.31+/-0.88, 4.38+/-0.25, and 5.45+/-1.78, respectively. These results suggest that, in addition to the specific chromosomal aberrations and cell death, elevated apoptosis could also be a marker for exposure to anthracene derivatives.     

Induction of apoptosis by cordycepin via reactive oxygen species generation in human leukemia cells

Cordycepin (3′-deoxyadenosin), a specific polyadenylation inhibitor, is the main functional component in Cordyceps militaris, one of the top three renowned traditional Chinese medicines. Cordycepin has been shown to possess many pharmacological activities including immunological stimulation, and anti-bacterial, anti-viral, and anti-tumor effects. However, the mechanisms underlying its anti-cancer mechanisms are not yet understood. In this study, the apoptotic effects of cordycepin were investigated in human leukemia cells. Treatment with cordycepin significantly inhibited cell growth in a concentration-dependent manner by inducing apoptosis but not necrosis. This induction was associated with generation of reactive oxygen species (ROS), mitochondrial dysfunction, activation of caspases, and cleavage of poly(ADP-ribose) polymerase protein. However, apoptosis induced by cordycepin was attenuated by caspase inhibitors, indicating an important role for caspases in cordycepin responses. Administration of N-acetyl-l-cysteine, a scavenger of ROS, also significantly inhibited cordycepin-induced apoptosis and activation of caspases. These results support a mechanism whereby cordycepin induces apoptosis of human leukemia cells through a signaling cascade involving a ROS-mediated caspase pathway.     

Prooxidant action of hinokitiol: hinokitiol-iron dependent generation of reactive oxygen species

Hinokitiol (alpha-thujaplicin, 2-hydroxy-4-isopropyl-2,4,6-cycloheptatrien-1-one), one of the tropolone compounds purified from the woods of Chamaecyparis and Thujopsis (hinoki and hiba), produced reactive oxygen species as a complex with transition metals. Hinokitiol/iron complex inactivated aconitase, the most sensitive enzyme to reactive oxygen, whereas it did not affect aldolase and glyceraldehyde 3-phosphate dehydrogenase. The inactivation of aconitase was iron-dependent, and prevented by TEMPOL, a scavenger of reactive oxygen species and superoxide dismutase, suggesting that the hinokitiol/iron-mediated generation of superoxide anion is responsible for the inactivation of aconitase. Addition of hinokitiol effectively enhanced the ascorbate/copper-mediated formation of 8-hydroxy-2'-deoxyguanosine in DNA. Cytotoxic effect of hinokitiol can be explained by its prooxidant properties: hinokitiol/transition metal complex generates reactive oxygen species causing inactivation of aconitase and production of hydroxyl radical resulting in the formation of DNA base adduct.