Reactive oxygen species in choline deficiency induced carcinogenesis and nitrone inhibition

Reactive oxygen species and free radical processes have been considered important in cancer development for many years. Much research demonstrates that the choline-deficiency induced hepatocarcinogenesis model prominently involves reactive oxygen species. We present a summary of results obtained in our original studies of this model over the last 4 years. We have shown that -phenyl-tert-butyl nitrone (PBN) and some of its hydroxylated derivatives (the 4- and 3-hydroxylated compounds) prevent hepatocarcinogenesis in this model. Mechanistic studies have demonstrated that isolated mitochondria from the livers of rats fed the choline-deficiency defined amino acid diet produce significantly much more H₂O₂ per NADH reducing equivalents oxidized. Based on these observations, we postulate that H₂O₂ is a primary carcinogenic factor in this model. Based on studies of the action of PBN on isolated mitochondria, we postulate that the inhibiting action of PBN involves suppression of H₂O₂ production of mitochondria and generally decreasing the oxidative stress within the preneoplastic lesions. The net effect of the activity of the nitrone compounds appears to be due to their ability to shift the apoptosis/neoplastic tendency balance toward apoptosis of the cells within the preneoplastic lesions. This is considered to be the primary reason the size of the preneoplastic lesions are significantly decreased and why the nitrones are potent anti-carcinogenic agents in this model.     

The nitrone spin trap PBN alters the cellular response to H(2)O(2): activation of the EGF receptor/ERK pathway

The nitrone spin trap PBN has been shown to protect neuronal cells from reactive oxygen species both in culture and in vivo. As an approach to understanding the molecular mechanisms by which PBN may function to protect cells, we examined whether PBN alters the cellular response to reactive oxygen species. H(2)O(2) stimulation of PC-12 cells results in weak activation of both the ERK and JNK signal transduction pathways. PBN pretreatment of PC-12 cells, followed by H(2)O(2) stimulation, results in strong and selective activation of the pro-survival ERK pathway. H(2)O(2) induction of ERK activity in PBN-pretreated cells was shown to be dependent on extracellular Ca(+2) influx. Further analysis of the ERK pathway showed that in PBN-pretreated cells, EGF receptor and the adapter protein SHC were phosphorylated in a Ca(+2)-dependent, ligand-independent manner following H(2)O(2) stimulation. Interestingly, H(2)O(2) stimulation of PBN-pretreated cells results in only 30% of the increase in intracellular Ca(+2) as compared to untreated cells following H(2)O(2) stimulation. These data suggest a model in which PBN attenuates H(2)O(2)-induced Ca(+2) entry, yet magnifies or alters Ca(+2) action, resulting in the activation of the EGF receptor/ERK pathway.     

Modulation of Fas-FasL related apoptosis by PBN in the early phases of choline deficient diet-mediated hepatocarcinogenesis in rats

This study focused on the detection of apoptosis related events in very early phases of choline-deficient (CD)-induced hepatocarcinogenesis (at 2–5 weeks). Flow cytometry of isolated intact primary hepatocytes from CD diet fed rats indicated increased expression of the apoptosis-associated protein Fas. Increased apoptosis in CD-treated livers was confirmed by Western blot analyses of caspases and cytochrome c. This study was also able to detect differences in apoptotic events following phenyl butyl nitrone (PBN) treatment. Fas expression was inhibited by PBN, indicating that PBN is anti-apoptotic. It is speculated that in the early stages of CD-induced hepatotoxicity, PBN is involved in inhibiting pro-inflammatory factor-driven apoptosis of normal hepatocytes, which protects against the initiation of carcinogenesis. The CD diet model is also considered as a model for non-alcoholic steatohepatitis (NASH) in humans and early expression of Fas could also be a good index of the progression of NASH.     

Modulation of Fas-FasL related apoptosis by PBN in the early phases of choline deficient diet-mediated hepatocarcinogenesis in rats

This study focused on the detection of apoptosis related events in very early phases of choline-deficient (CD)-induced hepatocarcinogenesis (at 2-5 weeks). Flow cytometry of isolated intact primary hepatocytes from CD diet fed rats indicated increased expression of the apoptosis-associated protein Fas. Increased apoptosis in CD-treated livers was confirmed by Western blot analyses of caspases and cytochrome c. This study was also able to detect differences in apoptotic events following phenyl butyl nitrone (PBN) treatment. Fas expression was inhibited by PBN, indicating that PBN is anti-apoptotic. It is speculated that in the early stages of CD-induced hepatotoxicity, PBN is involved in inhibiting pro-inflammatory factor-driven apoptosis of normal hepatocytes, which protects against the initiation of carcinogenesis. The CD diet model is also considered as a model for non-alcoholic steatohepatitis (NASH) in humans and early expression of Fas could also be a good index of the progression of NASH.     

Doxorubicin-induced apoptosis in endothelial cells and cardiomyocytes is ameliorated by nitrone spin traps and ebselen. Role of reactive oxygen and nitrogen species

Doxorubicin (DOX) is a broad spectrum anthracycline antibiotic used to treat a variety of cancers. Redox activation of DOX to form reactive oxygen species has been implicated in DOX-induced cardiotoxicity. In this work we investigated DOX-induced apoptosis in cultured bovine aortic endothelial cells and cardiomyocytes isolated from adult rat heart. Exposure of bovine aortic endothelial cells or myocytes to submicromolar levels of DOX induced significant apoptosis as measured by DNA fragmentation and terminal deoxynucleotidyltransferase-mediated nick-end labeling assays. Pretreatment of cells with 100 microm nitrone spin traps, N-tert-butyl-alpha-phenylnitrone (PBN) or alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) dramatically inhibited DOX-induced apoptosis. Ebselen (20-50 microm), a glutathione peroxidase mimetic, also significantly inhibited apoptosis. DOX (0.5-1 microm) inactivated mitochondrial complex I by a superoxide-dependent mechanism. PBN (100 microm), POBN (100 microm), and ebselen (50 microm) restored complex I activity. These compounds also inhibited DOX-induced caspase-3 activation and cytochrome c release. PBN and ebselen also restored glutathione levels in DOX-treated cells. We conclude that nitrone spin traps and ebselen inhibit the DOX-induced apoptotic signaling mechanism and that this antiapoptotic mechanism may be linked in part to the inhibition in formation or scavenging of hydrogen peroxide. Therapeutic strategies to mitigate DOX cardiotoxicity should be reexamined in light of these emerging antiapoptotic mechanisms of antioxidants.     

Comparison of the radical trapping ability of PBN, S-PPBN and NXY-059

The nitrones alpha-phenyl-N-tert-butyl nitrone (PBN), sodium 2-sulfophenyl-N-tert-butyl nitrone (S-PBN) and disodium 2,4-disulfophenyl-N-tert-butyl nitrone (NXY-059) are neuroprotective in a variety of rodent models. The objective of the current studies was to compare the ability of PBN, S-PBN, and NXY-059 to form radical adducts and to prevent salicylate oxidation in an aqueous system. For the electron spin resonance (ESR) studies, hydroxyl radicals were generated with ultraviolet (UV) light and hydrogen peroxide. Secondary radicals were then produced by the addition of methanol, ethanol, isopropanol, dimethylsulfoxide, tetrahydrofuran or 1,4-dioxane. In addition, competition spin trapping studies were performed using PBN-alpha-(13) C and either S-PBN or NXY-059. In the salicylate studies, PBN, S-PBN and NXY-059 were compared to a variety of other antioxidants and reference compounds (cysteine, glutathione, ascorbate, uric acid, Tempo, Trolox, and Tirilizad) for their ability to prevent 2,3- and 2,5-dihydroxybenzoic acid formation induced by hydroxyl radical generating systems. All 3 nitrones trapped carbon- and oxygen-centered radicals to produce ESR-detectable radical adducts. Each nitrone also prevented salicylate oxidation, with PBN being the most effective. The ability of these 3 nitrones to prevent salicylate oxidation resembled that of most of the other compounds tested.     

Phenyl-N-tert-butylnitrone demonstrates broad-spectrum inhibition of apoptosis-associated gene expression in endotoxin-treated rats

Systemic exposure to gram-negative bacterial substances such as lipopolysaccharide (LPS, or endotoxin) induces an uncontrolled, massive inflammatory reaction which culminates in multiple system organ failure and death. Septic shock often does not respond to corticosteroids; however, certain low-molecular-weight antioxidant compounds have been discovered to possess potent anti-inflammatory action, and some of these novel compounds can rescue animals from experimentally induced septic shock. Phenyl-N-tert-butylnitrone (PBN) is the archetype of the nitrone class of antioxidants which we have previously shown to suppress LPS-induced cytokine biosynthesis in vivo. Using a multiprobe ribonuclease protection assay, we now demonstrate the ability of PBN to suppress proapoptotic gene expression in the LPS-induced model of endotoxic shock. The broad-spectrum gene-suppressive affects of PBN are discussed in the context of inflammatory signal transduction and models are proposed to explain why certain antioxidants may also possess anti-inflammatory and antiapoptotic properties.     

Enhancement of apoptosis by nitric oxide released from alpha-phenyl-tert-butyl nitrone under hyperthermic conditions

The aim of this study was to examine whether a neuroprotector, PBN (alpha-phenyl-tert-butyl nitrone), enhances apoptosis induced by hyperthermia, which generates superoxide (O2-) intracellularly, since the release of nitric oxide (NO) from PBN under oxidative stress has been reported. When human myelomonocytic lymphoma U937 cells were treated with hyperthermia (44 degrees C, 10 min) and PBN, an increase in the concentration of nitrite in the culture medium, and a decrease in the hyperthermia-induced production of O2- was observed. Imaging using a fluorescence dye for intracellular NO, diaminofluorescein-2 diacetate (DAF-2 DA), revealed the formation of NO in the apoptotic cells treated with hyperthermia and PBN combined. Apoptotic endpoints were significantly enhanced by the combined treatment: a decrease in mitochondrial trans-membrane potential, cleavage of Bid, release of cytochrome c, and activation of caspase-8 and -3. An increase in the intracellular Ca2+ concentration ([Ca2+]i), externalization of Fas, and decrease in Hsp70 and phosphorylated HSF1 were observed following the combined treatment. Furthermore, scavengers of NO an d ONOO- significantly inhibited the enhancement of apoptosis, the externalization of Fas and the increase in [Ca2+]i. These results suggest that, (1) NO is released from PBN by hyperthermia, and subsequently reacts with O2- to form ONOO-, (2) NO and ONOO- are involved in the enhancement of apoptosis through Fas-mitochondria-caspase and [Ca2+]i-dependent pathways, and (3) a decrease in Hsp70 and phosphorylated HSF1 also contributed to the enhancement of apoptosis.     

Comparison of the radical trapping ability of PBN,S-PBN and NXY-059

The nitrones α-phenyl-N-tert-butyl nitrone (PBN), sodium 2-sulfophenyl-N-tert-butyl nitrone (S-PBN) and disodium 2,4-disulfophenyl-N-tert-butyl nitrone (NXY-059) are neuroprotective in a variety of rodent models. The objective of the current studies was to compare the ability of PBN, S-PBN, and NXY-059 to form radical adducts and to prevent salicylate oxidation in an aqueous system. For the electron spin resonance (ESR) studies, hydroxyl radicals were generated with ultraviolet (UV) light and hydrogen peroxide. Secondary radicals were then produced by the addition of methanol, ethanol, isopropanol, dimethylsulfoxide, tetrahydrofuran or 1,4-dioxane. In addition, competition spin trapping studies were performed using PBN-α-¹³C and either S-PBN or NXY-059. In the salicylate studies, PBN, S-PBN and NXY-059 were compared to a variety of other antioxidants and reference compounds (cysteine, glutathione, ascorbate, uric acid, Tempo, Trolox, and Tirilizad) for their ability to prevent 2,3- and 2,5-dihydroxybenzoic acid formation induced by hydroxyl radical generating systems. All 3 nitrones trapped carbon- and oxygen-centered radicals to produce ESR-detectable radical adducts. Each nitrone also prevented salicylate oxidation, with PBN being the most effective. The ability of these 3 nitrones to prevent salicylate oxidation resembled that of most of the other compounds tested.     

Apoptosis induced by dithiothreitol in HL-60 cells shows early activation of caspase 3 and is independent of mitochondria

Previous studies have shown that under certain conditions some thiol-containing compounds can cause apoptosis in a number of different cell lines. Herein, we investigated the apoptotic pathways in HL-60 cells triggered by dithiothreitol (DTT), used as a model thiol compound, and tested the hypothesis that thiols cause apoptosis via production of hydrogen peroxide (H2O2) during thiol oxidation. The results show that, unlike H2O2, DTT does not induce apoptosis via a mitochondrial pathway. This is demonstrated by the absence of early cytochrome c release from mitochondria into the cytosol, the lack of mitochondrial membrane depolarization at early times, and the minor role of caspase 9 in DTT-induced apoptosis. The first caspase activity detectable in DTT-treated cells is caspase 3, which is increased significantly 1 - 2 h after the start of DTT treatment. This was shown by following the cleavage of both a natural substrate, DFF-45/ICAD, and a synthetic fluorescent substrate, z-DEVD-AFC. Cleavage of substrates of caspases 2 and 8, known as initiator caspases, does not start until 3 - 4 h after DTT exposure, well after caspase 3 has become active and at a time when apoptosis is in late stages, as shown by the occurrence of DNA fragmentation to oligonucleosomal-sized pieces. Although oxidizing DTT can produce H2O2, data presented here indicate that DTT-induced apoptosis is not mediated by production of H2O2 and occurs via a novel pathway that involves activation of caspase 3 at early stages, prior to activation of the common 'initiator' caspases 2, 8 and 9.     

Hydrogen peroxide and redox modulation sensitize primary mouse hepatocytes to TNF-induced apoptosis

Tumor necrosis factor alpha (TNF) plays an important role in mediating hepatocyte injury in various liver pathologies. TNF treatment alone does not cause the death of primary cultured hepatocytes, suggesting other factors are necessary to mediate TNF-induced injury. In this work the question of whether reactive oxygen species can sensitize primary cultured hepatocytes to TNF-induced apoptosis and necrosis was investigated. Sublethal levels of H(2)O(2), either as bolus doses or steady-state levels generated by glucose oxidase, were found to sensitize cultured hepatocytes to TNF-induced apoptosis. High levels of H(2)O(2) also triggered necrosis in hepatocytes regardless of whether TNF was present. Similarly, antimycin, a complex III inhibitor that increases reactive oxygen species generation from mitochondria, sensitized hepatocytes to TNF-induced apoptosis at low doses but caused necrosis at high doses. Redox changes seem to be important in sensitizing primary hepatocytes, because diamide, a thiol-oxidizing agent, and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of GSSG reductase, also increased TNF-induced apoptosis in cultured primary hepatocytes at sublethal doses. High doses of diamide and BCNU predominantly triggered necrotic cell death. Agents that sensitized hepatocytes to TNF-induced apoptosis -- H(2)O(2), antimycin, diamide, BCNU -- all caused a dramatic fall in the GSH/GSSG ratio. These redox alterations were found to inhibit TNF-induced IkappaB-alpha phosphorylation and NF-kappaB translocation to the nucleus, thus presumably inhibiting expression of genes necessary to inhibit the cytotoxic effects of TNF. Taken together, these results suggest that oxidation of the intracellular environment of hepatocytes by reactive oxygen species or redox-modulating agents interferes with NF-kappaB signaling pathways to sensitize hepatocytes to TNF-induced apoptosis. The TNF-induced apoptosis seems to occur only in a certain redox range -- in which redox changes can inhibit NF-kappaB activity but not completely inhibit caspase activity. The implication for liver disease is that concomitant TNF exposure and reactive oxygen species, either extrinsically generated (e.g., nonparenchymal or inflammatory cells) or intrinsically generated in hepatocytes (e.g., mitochondria), may act in concert to promote apoptosis and liver injury.     

Contribution of mitochondrial DNA repair to cell resistance from oxidative stress

Numerous studies have revealed that a part of the cellular response to chronic oxidative stress involves increased antioxidant capacity. However, another defense mechanism that has received less attention is DNA repair. Because of the important homeostatic role of mitochondria and the exquisite sensitivity of mitochondrial DNA (mtDNA) to oxidative damage, we hypothesized that mtDNA repair plays an important role in the protection against oxidative stress. To test this hypothesis mtDNA damage and repair was evaluated in normal HA1 Chinese hamster fibroblasts and oxidative stress-resistant variants isolated following chronic exposure to H2O2 or 95% O2. Reactive oxygen species were generated enzymatically using xanthine oxidase and hypoxanthine. When treated with xanthine oxidase reduced levels of initial mtDNA damage and enhanced mtDNA repair were observed in the cells from the oxidative stress-resistant variants, relative to the parental cell line. This enhanced mtDNA repair correlated with an increase in mitochondrial apurinic/apyrimidinic endonuclease activity in both H2O2- and O2-resistant HA1 variants. This is the first report showing enhanced mtDNA repair in the cellular response to chronic oxidative stress. These results provide further evidence for the crucial role that mtDNA repair pathways play in protecting cells against the deleterious effects of reactive oxygen species.     

N-t-butyl hydroxylamine, a hydrolysis product of alpha-phenyl-N-t-butyl nitrone, is more potent in delaying senescence in human lung fibroblasts

Alpha-phenyl-N-t-butyl nitrone (PBN), a spin trap, scavenges hydroxyl radicals, protects tissues from oxidative injury, and delays senescence of both normal human lung fibroblasts (IMR90) and senescence-accelerated mice. N-t-butyl hydroxylamine and benzaldehyde are the breakdown products of PBN. N-t-Butyl hydroxylamine delays senescence of IMR90 cells at concentrations as low as 10 microM compared with 200 microM PBN to produce a similar effect, suggesting that N-t-butyl hydroxylamine is the active form of PBN. N-Benzyl hydroxylamine and N-methyl hydroxylamine compounds unrelated to PBN were also effective in delaying senescence, suggesting the active functional group is the N-hydroxylamine. All the N-hydroxylamines tested significantly decreased the endogenous production of oxidants, as measured by the oxidation of 2', 7'-dichlorodihydrofluorescin and the increase in the GSH/GSSG ratio. The acceleration of senescence induced by hydrogen peroxide is reversed by the N-hydroxylamines. DNA damage, as determined by the level of apurinic/apyrimidinic sites, also decreased significantly following treatment with N-hydroxylamines. The N-hydroxylamines appear to be effective through mitochondria; they delay age-dependent changes in mitochondria as measured by accumulation of rhodamine-123, they prevent reduction of cytochrome C(FeIII) by superoxide radical, and they reverse an age-dependent decay of mitochondrial aconitase, suggesting they react with the superoxide radical.     

Generation of hydrogen peroxide by brain mitochondria: the effect of reoxygenation following postdecapitative ischemia

The hypothesis that mitochondria damaged during complete cerebral ischemia generate increased amounts of superoxide anion radical and hydrogen peroxide (H2O2) upon postischemic reoxygenation has been tested. In rat brain mitochondria, succinate supported H2O2 generation, whereas NADH-linked substrates, malate plus glutamate, did so only in the presence of respiratory chain inhibitors. Succinate-supported H2O2 generation was diminished by rotenone and the uncoupler carbonyl cyanide m-chlorphenylhydrazone and enhanced by antimycin A and increased oxygen tensions. When maximally reduced, the NADH dehydrogenase and the ubiquinone-cytochrome b regions of the electron transport chain are sources of H2O2. These studies suggest that a significant portion of H2O2 generation in brain mitochondria proceeds via the transfer of reducing equivalents from ubiquinone to the NADH dehydrogenase portion of the electron transport chain. Succinate-supported H2O2 generation by mitochondria isolated from rat brain exposed to 15 min of postdecapitative ischemia was 90% lower than that of control preparations. The effect of varying oxygen tensions on H2O2 generation by postischemic mitochondrial preparations was negligible compared with the increased H2O2 generation measured in control preparations. Comparison of the effects of respiratory chain inhibitors and oxygen tension on succinate-supported H2O2 generation suggests that the ability for reversed electron transfer is impaired during ischemia. These data do not support the hypothesis that mitochondrial free radical generation increases during postischemic reoxygenation.     

Role of JNK translocation to mitochondria leading to inhibition of mitochondria bioenergetics in acetaminophen-induced liver injury

Previously, we demonstrated JNK plays a central role in acetaminophen (APAP)-induced liver injury (Gunawan, B. K., Liu, Z. X., Han, D., Hanawa, N., Gaarde, W. A., and Kaplowitz, N. (2006) Gastroenterology 131, 165-178). In this study, we examine the mechanism involved in activating JNK and explore the downstream targets of JNK important in promoting APAP-induced liver injury in vivo. JNK inhibitor (SP600125) was observed to significantly protect against APAP-induced liver injury. Increased mitochondria-derived reactive oxygen species were implicated in APAP-induced JNK activation based on the following: 1) mitochondrial GSH depletion (maximal at 2 h) caused increased H2O2 release from mitochondria, which preceded JNK activation (maximal at 4 h); 2) treatment of isolated hepatocytes with H2O2 or inhibitors (e.g. antimycin) that cause increased H2O2 release from mitochondria-activated JNK. An important downstream target of JNK following activation was mitochondria based on the following: 1) JNK translocated to mitochondria following activation; 2) JNK inhibitor treatment partially protected against a decline in mitochondria respiration caused by APAP treatment; and 3) addition of purified active JNK to mitochondria isolated from mice treated with APAP plus JNK inhibitor (mitochondria with severe GSH depletion, covalent binding) directly inhibited respiration. Cyclosporin A blocked the inhibitory effect of JNK on mitochondria respiration, suggesting JNK was directly inducing mitochondrial permeability transition in isolated mitochondria from mice treated with APAP plus JNK inhibitor. Addition of JNK to mitochondria isolated from control mice did not affect respiration. Our results suggests that APAP-induced liver injury involves JNK activation, due to increased reactive oxygen species generated by GSH-depleted mitochondria, and translocation of activated JNK to mitochondria where JNK induces mitochondrial permeability transition and inhibits mitochondria bioenergetics.     

Atypical effect of some spin trapping agents: reversible inhibition of acetylcholinesterase

N-tert-butyl-alpha-phenylnitrone (PBN), a widely used nitrone-based free radical trap was recently shown to prevent acetylcholinesterase (AChE) inhibitors induced muscle fasciculations and brain seizures while being ineffective against glutamergic or cholinergic receptor agonist induced seizures. In the present study we compared the effects on AChE activity of four free radical spin traps PBN, alpha-(4-pyridil-1)-N-tert-butyl nitrone (POBN), N-tert-butyl-alpha-(2-sulfophenyl)-nitrone (S-PBN) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). The kinetics of AChE inhibition were studied in vitro using a spectrophotometric kinetic assay with AChE from rat brain, diaphragm, electric eel and mouse brain. Spin trapping compounds S-PBN and DEPMPO, in concentrations up to 3 mM did not inhibit hydrolysis of ACh, while PBN and POBN inhibited hydrolysis of ACh in a reversible and concentration-dependent manner. Double reciprocal plots of the reaction velocity against varying ACh concentrations at each inhibitor concentration were linear and generally indicated mixed type inhibition. PBN was the most potent inhibitor of mouse AChE with Ki and Ki' of 0.58 and 2.99 mM, respectively, and the weakest inhibitor of electric eel AChE. In contrast, POBN showed the highest affinity for electric eel enzyme, with Ki and Ki' values of 1.065 and 3.15 mM, respectively. These findings suggest that the effect of PBN and POBN on AChE activity does not depend on trapping of damaging reactive oxygen and that in addition to their antioxidant action other pharmacological effects of these compounds should be considered when neuroprotective actions of PBN or POBN are investigated.     

Glucocorticoid-induced apoptosis of thymocytes: requirement of proteasome-dependent mitochondrial activity

Thymocytes undergo negative and positive selection during development in the thymus. During this selection process, the majority of thymocytes are eliminated by apoptosis through signaling via TCR or die by neglect, possibly mediated through glucocorticoids. In this study, we report that thymocytes require molecular oxygen to undergo apoptosis induced by dexamethasone (DEX), a synthetic glucocorticoid, and treatment with N-acetyl-L-cysteine (NAC), a thiol antioxidant, inhibits thymocyte apoptosis in vivo as well as ex vivo. We detected elevated intracellular levels of hydrogen peroxide (H(2)O(2)) during DEX-induced apoptosis, which is reduced by NAC treatment, indicating that the elevated levels of intracellular H(2)O(2) are proapoptotic. We also show that loss of mitochondrial membrane potential, cytochrome c release, as well as caspase-3 activation induced by DEX are attenuated by NAC treatment. We identified the production site for H(2)O(2) as the ubiquinone cycle at complex III of mitochondria by using various inhibitors of the mitochondrial electron transport chain, and we show that the cell death events mediated by mitochondria are also significantly reduced when the inhibitors were used. Through inhibition of the proteasome, we also show that the production of H(2)O(2) and the cell death events mediated by mitochondria are regulated by proteosomal activities in DEX-induced thymocyte apoptosis. We conclude that in DEX-treated thymocytes, the increased production of H(2)O(2) originates from mitochondria and is proapoptotic for cell death mediated by mitochondria. We also conclude that all the apoptotic events mediated by mitochondria are regulated by proteasomes.     

17beta-estradiol suppresses ROS-induced apoptosis of CHO cells through inhibition of lipid peroxidation-coupled membrane permeability transition

Oxidative stress-induced apoptotic cell death has been implicated to play a critical role in the mechanism of corpus luteum regression and follicular atresia. Recent studies suggests that reactive oxygen species (ROS) might play important roles in the regulation of luteal function. The present work describes the inhibitory effect of 17beta-estradiol (E2) on ROS-induced mitochondrial membrane permeability transition (MPT) and apoptosis of Chinese hamster ovary (CHO) cells. ROS generated by Fe2+ and H2O2 induced mitochondrial lipid peroxidation, depolarization, activation of caspase-3 and DNA fragmentation in CHO cells by some E2-inhibitable mechanism. E2 suppressed the Fe2+/H2O2-induced lipid peroxidation and MPT of isolated mitochondria that was characterized by cyclosporin A-inhibitable swelling, depolarization and cytochrome c release. Furthermore, E2 scavenged the xanthine oxidase generated ROS. These results suggests that Fe2+/H2O2 induced MPT and apoptosis of CHO cells by a mechanism that could be suppressed by antioxidant properties of E2.     

Effect of cytochrome c on the generation and elimination of O2*- and H2O2 in mitochondria

The primary recognized function of cytochrome c is to act as an electron carrier transferring electrons from complex III to complex IV in the respiratory chain of mitochondria. Recent studies on cell apoptosis reveal that cytochrome c is responsible for the programmed cell death when it is released from mitochondria to cytoplasm. In this study we present evidence showing that cytochrome c plays an antioxidative role by acting on the generation and elimination of O(2)(*) and H(2)O(2) in mitochondria. The O(2)(*) and H(2)O(2) generation in cytochrome c-depleted Keilin-Hartree heart muscle preparation (HMP) is 7-8 times higher than that in normal HMP. The reconstitution of cytochrome c to the cytochrome c-depleted HMP causes the O(2)(*) and H(2)O(2) generation to exponentially decrease. An alternative electron-leak pathway of the respiratory chain is suggested to explain how cytochrome c affects on the generation and elimination of O(2)(*) and H(2)O(2) in mitochondria. Enough cytochrome c in the respiratory chain is needed for keeping O(2)(*) and H(2)O(2) at a lower physiological level. A dramatic increase of O(2)(*) and H(2)O(2) generation occurs when cytochrome c is released from the respiratory chain. The burst of O(2)(*) and H(2)O(2), which happens at the same time as cytochrome c release from the respiratory chain, should have some role in the early stage of cell apoptosis.     

Effects of amyloid-beta peptides on hydrogen peroxide-metabolizing enzymes in rat brain in vivo

Amyloid-beta (Abeta) peptides are components of senile plaques initiating degeneration of brain neurons in Alzheimer's disease. They increase reactive oxygen species generation that may exceed the defensive capacity of cells. To test the hypothesis, this study investigated the in vivo effects of Abeta peptides on mitochondrial and non-mitochondrial enzymic sources of reactive oxygen species and antioxidant enzymes in rat brain. Continuous intracerebroventricular infusion of both Abeta(25-35) and Abeta(1-40) for up to 14 days stimulated the hydrogen peroxide (H2O2) generation in isolated neocortex mitochondria. Infusion of Abeta(1-40) led to an increase in Mn-superoxide dismutase activity and a decrease in activities of catalase and glutathione peroxidase in mitochondria, to elevation of activities of Cu,Zn-superoxide dismutase and aldehyde oxidase, forwarded the conversion of xanthine dehydrogenase to xanthine oxidase and corresponding increase in the rate of H2O2 formation in the cytosol. Thus, Abeta peptides increase H2O2-formation and H2O2-forming enzyme activities and inhibit H2O2-consuming enzyme activities in mitochondria and cytosol in vivo. These studies suggest that disbalance between H2O2-generating and H2O2-metabolizing enzyme activities can contribute to oxidative stress underlying neurodegeneration and neuronal death in Alzheimer's disease.