Decrease In Reactive Oxygen Species Research Articles

Mitochondria protection from hypoxia/reoxygenation injury with mitochondria heat shock protein 70 overexpression

The majority of mitochondrial proteins are encoded by nuclear genes and synthesized in the cytosol as preproteins containing a mitochondria import sequence. Preproteins traverse the outer mitochondrial membrane in an unfolded state and then translocate through the inner membrane into the matrix via import machinery that includes mitochondrial heat shock protein 70 (mtHSP70). Neonatal rat cardiac myocytes (NCM) infected with an adenoviral vector expressing mtHSP70 or an empty control (Adv(-)) for 48 h were submitted to 8 h of simulated ischemia (hypoxia) followed by 16 h of reperfusion (reoxygenation). Infection with mtHSP70 virus yielded an increase in mtHSP70 protein in NCM mitochondria compared with Adv(-) (P < 0.05). Cell viability after simulated ischemia/reperfusion (I/R) was decreased in both Adv(-) and mtHSP70 groups, relative to control (P < 0.05), but mtHSP70-infected NCM had enhanced viability after I/R relative to Adv-infected NCM (P < 0.05). Simulated I/R caused an increase in reactive oxygen species generation and lipid peroxidation in Adv-infected NCM (P < 0.05, for both) that was not observed in mtHSP70-infected NCM. Mitochondrial complex III and IV activities were greater in mtHSP70-infected NCM after simulated I/R compared with Adv(-) (P < 0.05 for both). After simulated I/R, ATP content increased in mtHSP70-infected NCM, compared with Adv(-) (P < 0.05). Apoptotic markers were decreased in mtHSP70-infected NCM compared with Adv(-) after simulated I/R (P < 0.05). These results indicate that overexpression of mtHSP70 protects the mitochondria against damage from simulated I/R that may be due to a decrease in reactive oxygen species leading to preservation of mitochondrial complex function activities and ATP formation.

Age related decrease of reactive oxygen species in neat semen of healthy fertile men

Although age-related changes in the male reproductive system are universally recognized, the question of declining fertility with age is controversial. Recently, an interest in the use of ROS as markers of male infertility has arisen since ROS is related to fertilization capacity. The objective of this study was to analyze the relationship between age and levels of seminal free radicals in men presenting for voluntary sterilization.

Thyroxine Ameliorates Oxidative Stress by Inducing Lipid Compositional Changes in Human Lens Epithelial Cells

Lipid saturation and sphingolipids make model membranes less susceptible to oxidation. A human lens epithelial cell line, HLE B-3, was treated with thyroxine to determine whether this treatment increases lipid saturation and membrane sphingolipids, as it does in other tissues, and if so, to see whether the treatment ameliorates the affects of lipid oxidation. One group of HLE B-3 cells was treated with thyroxine, and another group was not. Cells were then grown in a normoxic (20% O(2)), or hyperoxic (80% O(2)), atmosphere. Phospholipid composition was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and (31)P nuclear magnetic resonance spectroscopy. Cell viability was determined with a trypan blue dye assay. A chromogenic reagent was used to measure the secondary products of lipid oxidation. After 6 days of growth in a hyperoxic atmosphere, the thyroxine-treated cells were 20 times more viable than were the untreated cells. As a result of thyroxine treatment, the phosphatidylcholine (PC)-to-sphingolipid molar ratio decreased significantly (by 52%), and the PCs were eight times more unsaturated than were the sphingomyelins. The decrease in the amount of PCs coupled with a 33% decrease in the average unsaturation of the sphingolipids resulted in a phospholipid membrane with fewer double bonds. Products of lipid oxidation were three times higher in untreated cells after growth in a hyperoxic atmosphere than in untreated cells grown in a normoxic atmosphere. Thyroxine treatment reduced the amount of lipid oxidation products by approximately 60% compared with that in untreated cells. A 100% increase in cardiolipin with thyroxine treatment may contribute to a decrease in reactive oxygen species generated by the mitochondria. The total antioxidant power was not affected by thyroxine. Therefore, thyroxine-induced fluctuations in antioxidant levels are unlikely to influence increased cell viability and a concomitant decrease in the amount of lipid oxidation products in thyroxine-treated cells. The results support the idea that membranes containing more cardiolipin and more sphingolipids and having higher levels of saturation are more resistant to oxidation and protect cells from oxidative stress. Development of a therapy to increase sphingomyelins and lipid saturation in the lens may delay the onset of cataract.

Hepatoprotective effects of lupeol and mango pulp extract of carcinogen induced alteration in Swiss albino mice

Lupeol, a triterpene present in mango and other fruits, is known to exhibit a number of pharmacological properties including antioxidant, antilithiatic, and antidiabetic effects. In the present study, chemopreventive properties of lupeol and mango pulp extract (MPE) were evaluated against 7,12-dimethylbenz(a)anthracene (DMBA) induced alteration in liver of Swiss albino mice. Lupeol (25 mg/kg body weight, bw) or 1 mL of 20% w/v aqueous MPE/mouse were daily given once for 1 wk after a single dose of DMBA (50 mg/kg bw). Lupeol/MPE supplementation effectively influenced the DMBA induced oxidative stress, characterized by restored antioxidant enzyme activities and decrease in lipid peroxidation. A reduction of apoptotic cell population in the hypodiploid region was observed in lupeol and MPE supplemented animals. The inhibition of apoptosis was preceded by decrease in reactive oxygen species (ROS) level and restoration of mitochondrial transmembrane potential followed by decreased DNA fragmentation. In DMBA treated animals, downregulation of antiapoptotic Bcl-2 and upregulation of proapoptotic Bax and Caspase 3 in mouse liver was observed. These alterations were restored by lupeol/MPE, indicating inhibition of apoptosis. Thus, lupeol/MPE was found to be effective in combating oxidative stress induced cellular injury of mouse liver by modulating cell-growth regulators.

Growth-hormone-releasing peptide 6 (GHRP6) prevents oxidant cytotoxicity and reduces myocardial necrosis in a model of acute myocardial infarction

Therapies aimed at enhancing cardiomyocyte survival following myocardial injury are urgently required. As GHRP6 [GH (growth hormone)-releasing peptide 6] has been shown to stimulate GH secretion and has beneficial cardiovascular effects, the aim of the present study was to determine whether GHRP6 administration reduces myocardial infarct size following acute coronary occlusion in vivo. Female Cuban Creole pigs were anaesthetized, monitored and instrumented to ensure a complete sudden left circumflex artery occlusion for 1 h, followed by a 72 h reperfusion/survival period. Animals were screened clinically before surgery and assigned randomly to receive either GHRP6 (400 microg/kg of body weight) or normal saline. Hearts were processed, and the area at risk and the infarct size were determined. CK-MB (creatine kinase MB) and CRP (C-reactive protein) levels and pathological Q-wave-affected leads were analysed and compared. Evaluation of the myocardial effect of GHRP6 also included quantitative histopathology, local IGF-I (insulin-growth factor-I) expression and oxidative stress markers. GHRP6 treatment did not have any influence on mortality during surgery associated with rhythm and conductance disturbances during ischaemia. Infarct mass and thickness were reduced by 78% and 50% respectively, by GHRP6 compared with saline (P<0.01). More than 50% of the GHRP6-treated pigs did not exhibit pathogological Q waves in any of the ECG leads. Quantitative histopathology and CK-MB and CRP serum levels confirmed the reduction in GHRP6-mediated necrosis (all P<0.05). Levels of oxidative stress markers suggested that GHRP6 prevented myocardial injury via a decrease in reactive oxygen species and by the preservation of antioxidant defence systems (all P<0.05). Myocardial IGF-I transcription was not amplified by GHRP6 treatment compared with the increase induced by the ischaemic episode in relation to expression in intact hearts (P<0.01). In conclusion, GHRP6 exhibits antioxidant effects which may partially contribute to reduce myocardial ischaemic damage.

Slowing the deterioration of mango fruit during cold storage by pre-storage application of oxalic acid

SummaryThe effects of pre-storage application of oxalic acid (OA) on the incidence of decay and ripening in mango fruit, and its physiological effects on the peel and flesh of mango were investigated after mango fruit (Mangifera indica L. cv. Zill) were dipped in 5 mM OA for 10 min at 25ºC then stored at 14º ± 1ºC for 5 weeks. In addition, spore germination and mycelium growth of Colletotrichum gloeosporioides Penz. on potato dextrose agar (PDA) media containing different concentrations of OA were measured. Application of OA decreased the incidence of decay and delayed the ripening process in mango fruit during cold storage. However, there was no reduction in fruit quality, as judged by the soluble solids content (SSC) and titratable acidity (TA) of fruit ripened at 25ºC for 3 d, following 3 weeks of cold storage. Oxalic acid-treatment resulted in decreased lipoxygenase (LOX) activity and increased superoxide dismutase (SOD) activity in the peel, and increased ascorbate peroxidase (APX) activity in both the peel and the flesh, coincident with a decrease in reactive oxygen species (ROS).At or above 5 mM, OA with unaltered (natural) pH or after neutralisation, also inhibited C. gloeosporioides development in vitro. It is suggested that the physiological effects of OA, in decreasing LOX activity and enhancing defense against oxidation damage, contributed to delaying the ripening process in mango fruit during cold storage. Thus, pre-storage application of OA could be a promising method to suppress post-harvest deterioration and extend the shelf-life of refrigerated mango fruit, possibly due to a combination of its physiological effect in delaying the ripening process, coupled with an inhibition of the development of pathogens such as C. gloeosporioides.

A transient decrease in reactive oxygen species in roots leads to root hair deformation in the legume–rhizobia symbiosis

* A possible role for reactive oxygen species (ROS) in root hair deformation in response to Nod factor (NF) was investigated using Medicago truncatula nodulation mutants, and an inhibitor and precursors of ROS. * In wild-type roots, ROS efflux transiently decreased approximately 1 h after NF treatment. Transcript accumulation of two NADPH oxidase homologs, respiratory burst oxidase homolog 2 (MtRBOH2) and MtRBOH3, also transiently decreased at 1 h. However, in the nonnodulating mutant Nod factor perception (nfp), transcript accumulation did not change. * Exogenous application of ROS prevented root hair swelling and branching induced by NF. When accumulation of ROS was prevented by diphenylene iodonium (DPI), NF did not induce root hair branching. Root treatment with DPI alone reduced ROS efflux and induced root hair tip swelling. Transient treatment of roots with DPI mimicked NF treatment and resulted in root hair branching in the absence of NF. A transient DPI treatment did not induce root hair branching in the nonlegumes Arabidopsis thaliana and tomato (Lycopersicon esculentum). * The results suggest a role for the transient reduction of ROS accumulation in governing NF-induced root hair deformation in legumes.

Propriétés anti-inflammatoires de l'insuline chez les patients en réanimation

Critically ill patients requiring intensive care for more than a few days have a high risk of death. A tight control of glucose levels by intense insulin therapy reduced morbidity in critically ill patients. Insulin acts by two major molecular pathways: a. reduction of inflammation process induced by free fatty acids excess in tissues b. decrease of reactive oxygen species (ROS) production induced by hyperglycemia. By these actions, insulin preserve mitochondrial function, enhances adiponectin secretion and probably modulates AMP-activated protein kinase activity, which in turn depletes lipids depots in tissues and restores glucose uptake and oxidation. Furthermore, it has been recently established that insulin prevents microcirculation alteration and subsequent cellular hypoxia by reducing iNOS expression and activity in endothelium. So, beneficial effects of insulin in critically ill patients are dependent on metabolic and non-metabolic molecular pathways.

Genetic and chemical rescue of the Saccharomyces cerevisiae phenotype induced by mitochondrial DNA polymerase mutations associated with progressive external ophthalmoplegia in humans

The human POLG gene encodes the catalytic subunit of mitochondrial DNA polymerase gamma (pol gamma). Mutations in pol gamma are associated with a spectrum of disease phenotypes including autosomal dominant and recessive forms of progressive external ophthalmoplegia, spino-cerebellar ataxia and epilepsy, and Alpers-Huttenlocher hepatocerebral poliodystrophy. Multiple deletions, or depletion of mtDNA in affected tissues, are the molecular hallmarks of pol gamma mutations. To shed light on the pathogenic mechanisms leading to these phenotypes, we have introduced in MIP1, the yeast homologue of POLG, two mutations equivalent to the human Y955C and G268A mutations, which are associated with dominant and recessive PEO, respectively. Both mutations induced the generation of petite colonies, carrying either rearranged (rho-) or no (rho0) mtDNA. Mutations in genes that control the mitochondrial supply of deoxynucleotides (dNTP) affect the mtDNA integrity in both humans and yeast. To test whether the manipulation of the dNTP pool can modify the effects of pol gamma mutations in yeast, we have overexpressed a dNTP checkpoint enzyme, ribonucleotide reductase, RNR1, or deleted its inhibitor, SML1. In both mutant strains, the petite mutability was dramatically reduced. The same result was obtained by exposing the mutant strains to dihydrolipoic acid, an anti-oxidant agent. Therefore, an increase of the mitochondrial dNTP pool and/or a decrease of reactive oxygen species can prevent the mtDNA damage induced by pol gamma mutations in yeast and, possibly, in humans.

Down-regulation of transglutaminase II leads to impaired motility of cancer cells by inactivation of the protein kinase, Akt, and decrease of reactive oxygen species

We employed RNA interference to determine the role of tissue transglutaminase II (TGase II) in motility of cancer cells. Down-regulation of TGase II by small interfering RNA against TGase II impaired adhesion and motility of HeLa cells by decreasing phosphorylation of the protein kinase, Akt, and reactive oxygen species (ROS). Over-expression of TGase II showed opposite effects. These results suggest potential utility of TGase II for development of therapeutic anti cancer vaccine.

Rehabilitation Decreases Exercise-induced Oxidative Stress in Chronic Obstructive Pulmonary Disease

The effect of exercise at different intensities as well as the effect of intensive supervised pulmonary rehabilitation on oxidative stress were studied for chronic obstructive pulmonary disease (COPD). Eleven patients with COPD and 11 healthy age-matched control subjects performed a maximal and submaximal exercise cycle ergometry test at 60% of peak workload. Patients with COPD performed these tests before and after 8 wk of pulmonary rehabilitation. Measurements were done before, immediately after, and 4 h after both exercise tests. At rest, increased oxidative stress was observed in patients compared with control subjects, as measured by urinary malondialdehyde (MDA; p < 0.05) and hydrogen peroxide (H2O2) in breath condensate (p < 0.05). In healthy control subjects, a significant increase in urinary MDA was observed 4 h after both exercise tests (p = 0.05), whereas H2O2 significantly increased immediately after maximal exercise (p < 0.05). In patients with COPD, before rehabilitation, reactive oxygen species-induced DNA damage in peripheral blood mononuclear cells, urinary MDA, and plasma uric acid were significantly increased after both exercise tests (p < 0.05), whereas no significant increase was observed in plasma MDA. In contrast, exhaled H2O2 was only significantly increased after maximal exercise (p < 0.02). Although after rehabilitation peak workload was increased by 24%, a similar oxidative stress response was found. Remarkably, a decrease in reactive oxygen species-induced DNA damage was detected after exercise at submaximal intensity despite increased exercise duration of 73%. In summary, patients with COPD had increased pulmonary and systemic oxidative stress both at rest and induced by exercise. In addition, pulmonary rehabilitation increased exercise capacity and was associated with reduced exercise-induced oxidative stress.

Cancer-Related Anorexia/Cachexia Syndrome and Oxidative Stress: An Innovative Approach beyond Current Treatment

AbstractObjective: Cancer-related anorexia/cachexia syndrome and oxidative stress play a key role in the progression and outcome of neoplastic disease. Patients and Methods: On the basis of our previously published studies and clinical experience, we have developed an innovative approach consisting of diet with high polyphenol content (400 mg), p.o. pharmaconutritional support enriched with n − 3 fatty acids (eicosapentaenoic acid and docosahexaenoic acid) 2 cans (237 mL each) per day, medroxiprogesterone acetate 500 mg/d, antioxidant treatment with α-lipoic acid 300 mg/d plus carbocysteine lysine salt 2.7 g/d plus vitamin E 400 mg/d plus vitamin A 30,000 IU/d plus vitamin C 500 mg/d, and selective cyclooxygenase-2 inhibitor Celecoxib 200 mg/d. The treatment is administered for 16 weeks. The following variables are evaluated: (a) clinical variables (stage and Eastern Cooperative Oncology Group performance status); (b) nutritional variables (lean body mass, appetite, and resting energy expenditure); (c) laboratory variables (serum levels of proinflammatory cytokines, C-reactive protein, and leptin and blood levels of reactive oxygen species and antioxidant enzymes); and (d) quality of life variables (European Organization for Research and Treatment of Cancer QLQ-C30, EQ-5Dindex, and EQ-5DVAS). A phase II nonrandomized study has been designed to enroll 40 patients with advanced cancer at different sites with symptoms of cancer-related anorexia/cachexia syndrome and oxidative stress. Results: As of January 2004, 28 patients have been enrolled: 25 patients were evaluable and 14 of them have completed the treatment (20 patients have completed 2 months of treatment). As for clinical response, five patients improved, three patients remained unchanged, and six patients worsened. The Eastern Cooperative Oncology Group performance status (grade) 1 remained unchanged. As for nutritional/functional variables, the lean body mass increased significantly at 2 and 4 months. As for laboratory variables, reactive oxygen species decreased significantly and proinflammatory cytokines interleukin-6 and tumor necrosis factor-α decreased significantly. As for quality of life, it comprehensively improved after treatment. Conclusions: The treatment has been shown to be effective for clinical response, increase of lean body mass, decrease of reactive oxygen species and proinflammatory cytokines, and improvement of quality of life. The treatment has been shown to be safe with good compliance of patients. The study is in progress (14 further patients will be included).

Visible light affects chemiluminescence of carp (Cyprinus carpio) blood leukocytes.

Visible light irradiations at doses of 5 and 12 J/cm(2) were applied to carp buffy coat leukocytes. The leukocytes response was measured by a chemiluminescence (CL) assay as basal (spontaneous) bCL and Ca ionophore-induced stimulated CL (StCL). The irradiation caused a significant decrease in bCL in six out of 14 fish (susceptible fish) and rendered eight out of 14 fish unsusceptible. An inhibitory effect of light intensity dependence was more pronounced at 12 J/cm(2). Furthermore, this inhibitory effect of irradiation on bCL was found in fish which displayed higher (433 +/- 90 cpm/mL) pre-irradiation bCL, compared to unsusceptible subjects (88 +/- 30 cpm/mL, p < 0.05). Similar differences in the intensity of preirradiation StCL were found between these fish groups (13,053 +/- 5086 as compared to 1077 +/- 294, p = 0.03). Moreover, the time-to-peak of StCL was significantly shorter in susceptible fish, indicating their hyper-reactivity. These data show the inhibitory effect of visible light irradiation on blood leukocyte CL response in fish. These results suggest the prevention of host hyper-response which may occur under natural conditions of fish life. An Electron Paramagnetic Resonance (EPR) study of illuminated carp blood cells reveals the formation of Ascorbate free radicals (AFR) that may explain the decrease in reactive oxygen species (ROS) concentration following irradiation.

Bioenergetic remodeling during cellular differentiation: changes in cytochrome c oxidase regulation do not affect the metabolic phenotype.

Myogenesis induces mitochondrial proliferation, a decrease in reactive oxygen species (ROS) production, and an increased reliance upon oxidative phosphorylation. While muscles typically possess 20%-40% excess capacity of cytochrome c oxidase (COX), undifferentiated myoblasts have only 5%-20% of the mitochondrial content of myotubes and muscles. We used two muscle lines (C2C12, Sol8) and 3T3-L1 pre-adipocytes to examine if changes in COX regulation or activity with differentiation cause a shift in metabolic phenotype (i.e., more oxidative, less glycolytic, less ROS). COX activity in vivo can be suppressed by its inhibitor, nitric oxide, or sub-optimal substrate (cytochrome c) concentrations. Inhibition of nitric oxide synthase via L-NAME had no effect on the respiration of adherent undifferentiated cells, although it did stimulate respiration of myoblasts in suspension. While cytochrome c content increased during differentiation, there was no correlation with respiratory rate or reliance on oxidative metabolism. There was no correlation between COX specific activity and oxidative metabolism between cell type or in relation to differentiation. These studies show that, despite the very low activities of COX, undifferentiated myoblasts and pre-adipocytes possess a reserve of COX capacity and changes in COX with differentiation do not trigger the shift in metabolic phenotype.

Radiation-induced genomic instability and bystander effects: related inflammatory-type responses to radiation-induced stress and injury? A review

Purpose : To review studies of radiation responses in the haemopoietic system in the context of radiation-induced genomic instability, bystander effects and inflammatory-type processes. Results : There is considerable evidence that cells that themselves are not exposed to ionizing radiation but are the progeny of cells irradiated many cell divisions previously may express a high frequency of gene mutations, chromosomal aberrations and cell death. These effects are collectively known as radiation-induced genomic instability. A second untargeted effect results in non-irradiated cells exhibiting responses typically associated with direct radiation exposure but occurs as a consequence of contact with irradiated cells or by receiving soluble signals from irradiated cells. These effects are collectively known as radiation-induced bystander effects. Reported effects include increases or decreases in damage-inducible and stress-related proteins; increases or decreases in reactive oxygen species, cell death or cell proliferation, and induction of mutations and chromosome aberrations. This array of responses is reminiscent of effects mediated by cytokines and other similar regulatory factors that may involve, but do not necessarily require, gap junction-mediated transfer, have multiple inducers and a variety of context-dependent consequences in different cell systems. That chromosomal instability in haemopoietic cells can be induced by an indirect bystander-type mechanism both in vitro and in vivo provides a potential link between these two untargeted effects and there are radiation responses in vivo consistent with the microenvironment contributing secondary cell damage as a consequence of an inflammatory-type response to radiation-induced injury. Intercellular signalling, production of cytokines and free radicals are features of inflammatory responses that have the potential for both bystander-mediated and persisting damage as well as for conferring a predisposition to malignancy. The induction of bystander effects and instabilities may reflect interrelated aspects of a non-specific inflammatory-type response to radiation-induced stress and injury and be involved in a variety of the pathological consequences of radiation exposures.

N-Acetylcysteine Augments The Cellular Redox Changes and Cytotoxic Activity of Internalized Mycobacterium Bovis in Human Bladder Cancer Cells

We determined whether changes in cellular reactive oxygen species correlated with mycobacteria internalization and bladder cancer cell death. Reactive oxygen species and thiols in RT112 and MGH bladder cancer cells were determined using the fluorescence probes 5-(and 6)-carboxy-2', 7' dichlorodihydrofluorescein diacetate and monobromobimane. Superoxide and nitrite production were measured using bis-N-methylarcridinium nitrate and Griess reagents. Cytotoxicity was determined by the release of 14C-thymidine from cells with 14C labeled DNA. MGH cells that internalize bacillus Calmette-Guerin (BCG) had decreased cellular reactive oxygen species and thiols, although superoxide and nitric oxide production increased. RT112 cells, which do not internalize BCG, did not show a decrease in reactive oxygen species after incubation with BCG. Blocking BCG uptake in MGH cells abrogated reactive oxygen species reduction, confirming that the changes in reactive oxygen species were internalization dependent events. Treating cells with BCG and the antioxidant N-acetylcysteine caused a greater reduction in reactive oxygen species, and induced earlier and greater cytotoxicity in MGH but not in RT112 cells. The induction of bladder cancer cell killing by BCG parallels the ability of cells to internalize BCG, which in turn indicates that the susceptibility of tumor cells to the cytotoxic effects of BCG may be related to changes in cellular levels of reactive oxygen species and thiols. Supplementation with an antioxidant could enhance the antitumor effect of BCG.

Wogonin and fisetin induce apoptosis in human promyeloleukemic cells, accompanied by a decrease of reactive oxygen species, and activation of caspase 3 and Ca 2+-dependent endonuclease

Seven structurally related flavonoids including luteolin, nobiletin, wogonin, baicalein, apigenin, myricetin and fisetin were used to study their biological activities on the human leukemia cell line, HL-60. On MTT assay, wogonin, baicalein, apigenin, myricetin and fisetin showed obvious cytotoxic effects on HL-60 cells, with wogonin and fisetin being the most-potent apoptotic inducers among them. The cytotoxic effects of wogonin and fisetin were accompanied by the dose- and time-dependent appearance of characteristics of apoptosis including DNA fragmentation, apoptotic bodies and the sub-G1 ratio. Treatment with an apoptosis-inducing concentration of wogonin or fisetin causes rapid and transient induction of caspase 3/CPP32 activity, but not caspase 1 activity. Further, cleavage of poly(ADP-ribose) polymerase (PARP) and decrease of pro-caspase 3 protein were detected in wogonin- and fisetin-treated HL-60 cells. An increase in the pro-apoptotic protein, bax, and a decrease in the anti-apoptotic protein, Mcl-1, were detected in fisetin- and wogonin-treated HL-60 cells. However, Bcl-2, Bcl-XL, and Bad all remained unchanged in wogonin- and fisetin-treated HL-60 cells. In vitro chromatin digestion revealed that endonuclease activity was profoundly enhanced in wogonin- and fisetin-treated HL-60 cells, and the addition of ethylenediaminetetraacetic acid (EDTA) or ethyleneglycoltetraacetic acid (EGTA) into the reaction blocked endonuclease activation and at an optimum pH of 7.5. The caspase 3 inhibitor, Ac-DEVD-CHO, but not the caspase 1 inhibitor, Ac-YVAD-CHO, attenuated wogonin- and fisetin-induced DNA ladders, PARP cleavage, and endonuclease activation. Pretreatment of HL-60 cells with N-acetyl-cysteine or catalase efficiently inhibited H 2O 2 (200 μM)-induced apoptosis, but showed no inhibitory effect on wogonin- and fisetin-induced DNA ladders, caspase 3 activation, or bax protein induction. Decrease in endogenous ROS production was detected in wogonin- and fisetin-treated HL-60 cells by DCHF-DA assay. In conclusion, our experiments indicate that a decrease in intracellular peroxide level was involved in wogonin- and fisetin-induced apoptosis; activation of caspase 3 and endonuclease, induction of bax protein and suppression of Mcl-1 protein were detected in the process.

The inhibitory effect of zinc on cadmium-induced cell apoptosis and reactive oxygen species (ROS) production in cell cultures

The prevention of apoptosis by Zn 2+ is a well-known phenomenon. Both in in vitro and in vivo Zn 2+ supplementation prevents apoptosis induced by a variety of agents, among them by cadmium ions. The target for protective action of Zn ions on cell apoptosis is still unknown. In this paper we have evaluated the effect of in vitro ZnCl 2 supplementation at a concentration corresponding to the physiological level (10 μM) and higher (50 μM), on apoptosis induced with different Cd concentrations in two cell types: HeLa human tumor cell line and bovine aorta endothelial cells (BAECs). We demonstrated that Zn supplementation, especially at 10 μM concentration, significantly inhibited apoptosis in both types of cells. To assess the mechanism involved in the Zn effect we examined the influence of Zn supplementation on Cd accumulation in cells, Cd-induced superoxide anion (O 2 −) and hydrogen peroxide (H 2O 2) production. Zn caused 1.2–2.0-fold inhibition of Cd accumulation, 1.2–2.0-fold inhibition of Cd-induced apoptotic cell death, 1.1–2.0-fold decrease in reactive oxygen species (ROS) production in HeLa cells and in BAECs. These results indicate that inhibition of Cd-induced apoptosis in cells by Zn might be due, not only by inhibition of Cd accumulation in cells but, at least in part, to inhibition of Cd-induced production of ROS, which in turn are known as strong inducers of apoptosis.

Possible role of ROS as mediators of hypoxia-induced ion transport inhibition of alveolar epithelial cells

In oxygen-sensitive excitable cells, responses to hypoxia are initiated by membrane depolarization due to closing of the K channels that is thought to be mediated by a decrease in reactive oxygen species (ROS). Because the mechanisms of hypoxic inhibition of ion transport of alveolar epithelial cells (Planes C, Friedlander G, Loiseau A, Amiel C, and Clerici C. Am J Physiol Lung Cell Mol Physiol 271: L70-L78, 1996; Mairbäurl H, Wodopia R, Eckes S, Schulz S, and Bärtsch P. Am J Physiol Lung Cell Mol Physiol 273: L797-L806, 1997) are not yet understood, we tested the possible involvement of a hypoxia-induced change in ROS that might control transport activity. Transport was measured as (86)Rb and (22)Na uptake in A549 cells exposed to normoxia, hyperoxia, or hypoxia together with ROS donors and scavengers. H(2)O(2) < 1 mM did not affect transport, whereas 1 mM H(2)O(2) activated (22)Na uptake (+200%) but inhibited (86)Rb uptake (-30%). Also hyperoxia, aminotriazole plus menadione, and diethyldithiocarbamate inhibited (86)Rb uptake. N-acetyl-L-cysteine, diphenyleneiodonium, and tetramethylpiperidine-N-oxyl, used to reduce ROS, inhibited (86)Rb uptake, thus mimicking the hypoxic effects, whereas deferoxamine, superoxide dismutase, and catalase were ineffective. Also, hypoxic effects on ion transport were not prevented in the presence of H(2)O(2), diethyldithiocarbamate, and N-acetyl-L-cysteine. These results indicate that ion transport of A549 cells is significantly affected by decreasing or increasing cellular ROS levels and that it is possible that certain species of ROS might mediate the hypoxic effects on ion transport of alveolar epithelial cells.

P53-Independent Inhibition of Proliferation and p21Waf1/Cip1-Modulated Induction of Cell Death by the Antioxidants N-acetylcysteine and Vitamin E

Epidemiological evidence has suggested an association between diets rich in antioxidants and diminished risks of various types of cancer. Proposed mechanisms for protective effects of antioxidants have involved inhibition of free radical-mediated DNA damage. Recent data suggest that antioxidants may prevent or eliminate cancerous cells through their ability to inhibit proliferation or to induce programmed cell death (PCD). To begin to identify cell cycle and cell death regulatory factors involved in antioxidant-induced growth arrest and PCD, we have studied colorectal carcinoma cells (CRCs) that differ in expression of the tumor suppressor protein p53, of the cyclindependent kinase (CDK) inhibitor p21Waf1/Cip1. The antioxidants, N-acetylcysteine (NAC) and vitamin E either inhibited proliferation in a p53-independent manner without affecting cell viability or induced cell death. Growth arrest was not associated with upregulation of the CDK inhibitors p21Waf1/Cip1, p18ink4c or p16ink4c, but was associated with a decrease in reactive oxygen species (ROS). In contrast to previous observations, the absence of p21Waf1/Cip1 increased susceptibility of CRCs to antioxidant-induced PCD. NAC decreased levels of retinoblastoma protein (Rb) phosphorylation in all cells tested, but Rb was cleaved only in cells which underwent NAC-induced death. Although NAC decreased ROS in all cells studied, cell lines in which PCD occurred had higher baseline levels of ROS than cell lines in which proliferation was blocked. These observations suggest that expression of p21Waf1/Cip1 and basal levels of ROS are important determinants of outcome after antioxidant treatment.