naphthoquinones and 2-3-dimethoxy-1-4-naphthoquinone

Topics: Animals; Cytochrome-B(5) Reductase; Dicumarol; Enzyme Inhibitors; gamma-Glutamyltransferase; Gene Expression Regulation, Enzymologic; Glutamate-Cysteine Ligase; Glutathione; Glutathione Peroxidase; Humans; Hydrogen Peroxide; Molecular Structure; NAD(P)H Dehydrogenase (Quinone); NADPH-Ferrihemoprotein Reductase; Naphthoquinones; Nitric Oxide Synthase; Oxidation-Reduction; Protein Isoforms; Quinone Reductases; Quinones; Response Elements; Trans-Activators; Transcription, Genetic; Tumor Cells, Cultured; Vitamin K 3; Xenobiotics

Signaling pathways controlling necrosis are still mysterious and debated. We applied a shRNA-based viability screen to identify critical elements of the necrotic response. We took advantage from a small molecule (G5) that makes covalent adducts with free thiols by Michael addition and elicits multiple stresses. In cells resistant to apoptosis, G5 triggers necrosis through the induction of protein unfolding, glutathione depletion, ER stress, proteasomal impairments, and cytoskeletal stress. The kinase GSK3β was isolated among the top hits of the screening. Using the quinone DMNQ, a ROS generator, we demonstrate that GSK3β is involved in the regulation of ROS-dependent necrosis. Our results have been validated using siRNA and by knocking-out GSK3β with the CRISPR/Cas9 technology. In response to DMNQ GSK3β is activated by serine 9 dephosphorylation, concomitantly to Akt inactivation. During the quinone-induced pro-necrotic stress, GSK3β gradually accumulates into the nucleus, before the collapse of the mitochondrial membrane potential. Accumulation of ROS in response to DMNQ is impaired by the absence of GSK3β. We provide evidence that the activities of the obligatory two-electrons reducing flavoenzymes, NQO1 (NAD(P)H quinone dehydrogenase 1) and NQO2 are required to suppress DMNQ-induced necrosis. In the absence of GSK3β the expression of NQO1 and NQO2 is dramatically increased, possibly because of an increased transcriptional activity of NRF2. In summary, GSK3β by blunting the anti-oxidant response and particularly NQO1 and NQO2 expression, favors the appearance of necrosis in response to ROS, as generated by the quinone DMNQ.

Topics: Cell Line; Cell Nucleus; Enzyme Activation; Genetic Testing; Glycogen Synthase Kinase 3 beta; Green Fluorescent Proteins; Humans; Membrane Potential, Mitochondrial; Mitochondria; Models, Biological; Naphthoquinones; Necroptosis; NF-E2-Related Factor 2; Oxidative Stress; Reactive Oxygen Species; Reproducibility of Results; RNA, Small Interfering

The cytoprotective mechanism of l-serine against oxidative stress-mediated neuronal apoptosis was investigated in mouse hippocampal neuronal HT22 cells. Treatment with the reactive oxygen species (ROS) inducer 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) increased cytosolic and mitochondrial ROS and apoptosis, without necrosis, in HT22 cells. ROS-mediated apoptosis was accompanied by the induction of the endoplasmic reticulum (ER) stress-mediated apoptotic pathway, involving CHOP/GADD153 upregulation, JNK and p38 MAPK activation, and caspase-12 and caspase-8 activation, and subsequent induction of the mitochondrial apoptotic pathway through BAK and BAX activation, mitochondrial membrane potential (Δψm) loss, caspase-9 and caspase-3 activation, PARP cleavage, and nucleosomal DNA fragmentation. However, the DMNQ-caused ROS elevation and ER stress- and mitochondrial damage-induced apoptotic events were dose-dependently suppressed by co-treatment with l-serine (7.5-20 mM). Although DMNQ reduced both the intracellular glutathione (GSH) level and the ratios of reduced GSH to oxidized GSH (GSSG), the reduction was restored by co-treatment with l-serine. Co-treatment with GSH or N-acetylcysteine also blocked DMNQ-caused ROS elevation and apoptosis; however, co-treatment with the GSH synthesis inhibitor buthionine sulfoximine significantly promoted ROS-mediated apoptosis and counteracted the protection by l-serine. In HT22 cells, DMNQ treatment appeared to tilt the mitochondrial fusion-fission balance toward fission by down-regulating the levels of profusion proteins (MFN1/2 and OPA1) and inhibitory phosphorylation of profission protein DRP1 at Ser-637, resulting in mitochondrial fragmentation. These DMNQ-caused alterations were prevented by l-serine. A comparison of mitochondrial energetic function between DMNQ- and DMNQ/l-serine-treated HT22 cells showed that the DMNQ-caused impairment of the mitochondrial energy generation capacity was restored by l-serine. These results demonstrate that l-serine can protect neuronal cells against oxidative stress-mediated apoptotic cell death by contributing to intracellular antioxidant GSH synthesis and maintaining the mitochondrial fusion-fission balance.

Topics: Animals; Antioxidants; Apoptosis; Cell Line; Endoplasmic Reticulum Stress; Hippocampus; Membrane Potential, Mitochondrial; Mice; Mitochondria; Naphthoquinones; Neurons; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protective Agents; Reactive Oxygen Species; Serine

Metabolic and bioenergetic dysfunction are associated with oxidative stress and thought to be a common underlying mechanism of chronic diseases such as atherosclerosis, diabetes, and neurodegeneration. Recent findings support an emerging concept that circulating leukocytes and platelets can act as sensors or biomarkers of mitochondrial function in patients subjected to metabolic diseases. It is proposed that systemic stress-induced alterations in leukocyte bioenergetics are the consequence of several factors including reactive oxygen species. This suggests that oxidative stress mediated changes in leukocyte mitochondrial function could be used as an indicator of bioenergetic health in individuals. To test this concept, we investigated the effect of the redox cycling agent, 2,3 dimethoxynaphthoquinone (DMNQ) on the bioenergetic profiles of monocytes isolated from healthy human subjects using the extracellular flux analyzer. In addition, we tested the hypothesis that the bioenergetic health index (BHI), a single value that represents the bioenergetic health of individuals, is dynamically sensitive to oxidative stress in human monocytes. DMNQ decreased monocyte ATP-linked respiration, maximal respiration, and reserve capacity and caused an increase in proton leak and non-mitochondrial respiration compared to monocytes not treated with DMNQ. The BHI was a more sensitive indicator of the DMNQ-dependent changes in bioenergetics than any individual parameter. These data suggest that monocytes are susceptible to oxidative stress mediated by DMNQ and this can be accurately assessed by the BHI. Taken together, our findings suggest that the BHI has the potential to act as a functional biomarker of the impact of systemic oxidative stress in patients with metabolic disorders.

Topics: Adult; Biomarkers; Energy Metabolism; Female; Humans; Male; Mitochondria; Monocytes; Naphthoquinones; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species

Aberrant epigenetic modifications are implicated in maternal diabetes-induced neural tube defects (NTDs). Because cellular stress plays a causal role in diabetic embryopathy, we investigated the possible role of the stress-resistant sirtuin (SIRT) family histone deacetylases. Among the seven sirtuins (SIRT1-7), pre-gestational maternal diabetes in vivo or high glucose in vitro significantly reduced the expression of SIRT 2 and SIRT6 in the embryo or neural stem cells, respectively. The down-regulation of SIRT2 and SIRT6 was reversed by superoxide dismutase 1 (SOD1) over-expression in the in vivo mouse model of diabetic embryopathy and the SOD mimetic, tempol and cell permeable SOD, PEGSOD in neural stem cell cultures. 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), a superoxide generating agent, mimicked high glucose-suppressed SIRT2 and SIRT6 expression. The acetylation of histone 3 at lysine residues 56 (H3K56), H3K14, H3K9, and H3K27, putative substrates of SIRT2 and SIRT6, was increased by maternal diabetes in vivo or high glucose in vitro, and these increases were blocked by SOD1 over-expression or tempol treatment. SIRT2 or SIRT6 over-expression abrogated high glucose-suppressed SIRT2 or SIRT6 expression, and prevented the increase in acetylation of their histone substrates. The potent sirtuin activator (SRT1720) blocked high glucose-increased histone acetylation and NTD formation, whereas the combination of a pharmacological SIRT2 inhibitor and a pan SIRT inhibitor mimicked the effect of high glucose on increased histone acetylation and NTD induction. Thus, diabetes in vivo or high glucose in vitro suppresses SIRT2 and SIRT6 expression through oxidative stress, and sirtuin down-regulation-induced histone acetylation may be involved in diabetes-induced NTDs. The mechanism underlying pre-gestational diabetes-induced neural tube defects (NTDs) is still elusive. Our study unravels a new epigenetic mechanism in which maternal diabetes-induced oxidative stress represses sirtuin deacetylase 2 (SIRT2) and 6 (SIRT6) expression leading to histone acetylation and gene expression. SIRT down-regulation mediates the teratogenicity of diabetes leading to (NTD) formation. The study provides a mechanistic basis for the development of natural antioxidants and SIRT activators as therapeutics for diabetic embryopathy.

Topics: Acetylation; Animals; Diabetes, Gestational; Epigenesis, Genetic; Female; Glucose; Group III Histone Deacetylases; Heterocyclic Compounds, 4 or More Rings; Histones; Mice; Naphthoquinones; Neural Stem Cells; Neural Tube Defects; Oxidative Stress; Pregnancy; Sirtuin 2; Sirtuins; Superoxide Dismutase; Superoxide Dismutase-1

Our recent studies indicate that hydrogen peroxide (H2O2) only at high concentrations can cause oxidative stress in renal epithelial cells and induce apoptosis of podocytes. Consistently, the present study shows that H2O2, even at 1 mM, failed to induce intracellular oxidative stress and apoptosis of the podocytes due to efficient activity of catalase, an enzyme which degrades H2O2 to produce water and oxygen (O2). However, H2O2 acted as a source of O2 to allow acute ethanol to induce superoxide production and cause apoptosis of the podocytes. In contrast, acute ethanol alone did not elevate intracellular superoxide, even though it stimulates expression and translocation of p47phox to the plasma membrane. Inhibition of catalase abolished not only O2 production from H2O2 degradation, but also NOX2-dependent superoxide production in the podocytes challenged by both H2O2 and acute ethanol. In parallel, acute ethanol in the presence of H2O2, but neither ethanol nor H2O2 alone, stimulated transient receptor potential canonical 6 (TRPC6) channels and caused TRPC6-dependent elevation of intracellular Ca2+. These data suggest that exogenous H2O2 does not induce oxidative stress due to rapid degradation to produce O2 in the podocytes, but the oxygenated podocytes become sensitive to acute ethanol challenge and undergo apoptosis via a TRPC6-dependent elevation of intracellular Ca2+. Since cultured podocytes are considered in hypoxic conditions, H2O2 may be used as a source of O2 to establish an ischemia-reperfusion model in some type of cultured cells in which H2O2 does not directly induce intracellular oxidative stress.

Topics: Apoptosis; Catalase; Cell Line; Cell Membrane; Ethanol; Humans; Hydrogen Peroxide; Intracellular Space; Ion Channel Gating; Membrane Glycoproteins; NADPH Oxidase 2; NADPH Oxidases; Naphthoquinones; Oxidative Stress; Oxygen; Podocytes; Protein Transport; Superoxides; TRPC Cation Channels; TRPC6 Cation Channel

Activation of the phagocytic NADPH oxidase-2 (NOX-2) in neutrophils is a critical process in the innate immune system and is associated with elevated local concentrations of superoxide, hydrogen peroxide (H2O2) and hypochlorous acid. Under pathological conditions, NOX-2 activity has been implicated in the development of autoimmunity, indicating a role in modulating lymphocyte effector function. Notably, T-cell clonal expansion and subsequent cytokine production requires a metabolic switch from mitochondrial respiration to aerobic glycolysis. Previous studies demonstrate that H2O2 generated from activated neutrophils suppresses lymphocyte activation but the mechanism is unknown. We hypothesized that activated neutrophils would prevent the metabolic switch and suppress the effector functions of T-cells through a H2O2-dependent mechanism. To test this, we developed a model co-culture system using freshly isolated neutrophils and lymphocytes from healthy human donors. Extracellular flux analysis was used to assess mitochondrial and glycolytic activity and FACS analysis to assess immune function. The neutrophil oxidative burst significantly inhibited the induction of lymphocyte aerobic glycolysis, caused inhibition of oxidative phosphorylation and suppressed lymphocyte activation through a H2O2-dependent mechanism. Hydrogen peroxide and a redox cycling agent, DMNQ, were used to confirm the impact of H2O2 on lymphocyte bioenergetics. In summary, we have shown that the lymphocyte metabolic switch from mitochondrial respiration to glycolysis is prevented by the oxidative burst of neutrophils. This direct inhibition of the metabolic switch is then a likely mechanism underlying the neutrophil-dependent suppression of T-cell effector function.

Topics: Cells, Cultured; Coculture Techniques; Glycolysis; Humans; Hydrogen Peroxide; Immunity, Innate; Lymphocytes; Naphthoquinones; Neutrophil Activation; Neutrophils; Oxidation-Reduction; Peroxidase; Respiratory Burst; Superoxides; Suppressor Factors, Immunologic; T-Lymphocyte Subsets

There is increasing recognition that mitochondrial dysfunction is associated with the autism spectrum disorders. However, little attention has been given to the etiology of mitochondrial dysfunction or how mitochondrial abnormalities might interact with other physiological disturbances associated with autism, such as oxidative stress. In the current study we used respirometry to examine reserve capacity, a measure of the mitochondrial ability to respond to physiological stress, in lymphoblastoid cell lines (LCLs) derived from children with autistic disorder (AD) as well as age and gender-matched control LCLs. We demonstrate, for the first time, that LCLs derived from children with AD have an abnormal mitochondrial reserve capacity before and after exposure to increasingly higher concentrations of 2,3-dimethoxy-1,4-napthoquinone (DMNQ), an agent that increases intracellular reactive oxygen species (ROS). Specifically, the AD LCLs exhibit a higher reserve capacity at baseline and a sharper depletion of reserve capacity when ROS exposure is increased, as compared to control LCLs. Detailed investigation indicated that reserve capacity abnormalities seen in AD LCLs were the result of higher ATP-linked respiration and maximal respiratory capacity at baseline combined with a marked increase in proton leak respiration as ROS was increased. We further demonstrate that these reserve capacity abnormalities are driven by a subgroup of eight (32%) of 25 AD LCLs. Additional investigation of this subgroup of AD LCLs with reserve capacity abnormalities revealed that it demonstrated a greater reliance on glycolysis and on uncoupling protein 2 to regulate oxidative stress at the inner mitochondria membrane. This study suggests that a significant subgroup of AD children may have alterations in mitochondrial function which could render them more vulnerable to a pro-oxidant microenvironment derived from intrinsic and extrinsic sources of ROS such as immune activation and pro-oxidant environmental toxicants. These findings are consistent with the notion that AD is caused by a combination of genetic and environmental factors.

Topics: Autistic Disorder; Case-Control Studies; Cell Line; Cell Respiration; Child; Child, Preschool; Female; Glycolysis; Humans; Ion Channels; Lymphocytes; Male; Mitochondria; Mitochondrial Membranes; Mitochondrial Proteins; Naphthoquinones; Oxidation-Reduction; Oxidative Stress; Protons; Reactive Oxygen Species; Uncoupling Protein 2

Psoriasis is a chronic inflammatory skin disease resulting from immune dysregulation. Regulatory T cells (Tregs) are important in the prevention of psoriasis. Traditionally, reactive oxygen species (ROS) are known to be implicated in the progression of inflammatory diseases, including psoriasis, but many recent studies suggested the protective role of ROS in immune-mediated diseases. In particular, severe cases of psoriasis vulgaris have been reported to be successfully treated by hyperbaric oxygen therapy (HBOT), which raises tissue level of ROS. Also it was reported that Treg function was closely associated with ROS level. However, it has been only investigated in lowered levels of ROS so far. Thus, in this study, to clarify the relationship between ROS level and Treg function, as well as their role in the pathogenesis of psoriasis, we investigated imiquimod-induced psoriatic dermatitis (PD) in association with Treg function both in elevated and lowered levels of ROS by using knockout mice, such as glutathione peroxidase-1(-/-) and neutrophil cytosolic factor-1(-/-) mice, as well as by using HBOT or chemicals, such as 2,3-dimethoxy-1,4-naphthoquinone and N-acetylcysteine. The results consistently showed Tregs were hyperfunctional in elevated levels of ROS, whereas hypofunctional in lowered levels of ROS. In addition, imiquimod-induced PD was attenuated in elevated levels of ROS, whereas aggravated in lowered levels of ROS. For the molecular mechanism that may link ROS level and Treg function, we investigated the expression of an immunoregulatory enzyme, indoleamine 2,3-dioxygenase (IDO) which is induced by ROS, in PD lesions. Taken together, it was implied that appropriately elevated levels of ROS might prevent psoriasis through enhancing IDO expression and Treg function.

Topics: Acetylcysteine; Aminoquinolines; Animals; Dermatitis; Disease Progression; Glutathione Peroxidase; Glutathione Peroxidase GPX1; Hyperbaric Oxygenation; Imiquimod; Indoleamine-Pyrrole 2,3,-Dioxygenase; Mice, Inbred C57BL; NADPH Oxidases; Naphthoquinones; Psoriasis; Reactive Oxygen Species; T-Lymphocytes, Regulatory

Apoptosis of vascular smooth muscle cells (VSMC) significantly contributes to the instability of advanced atherosclerotic plaques. Oxygen radicals are an important cause for VSMC death. However, the precise mechanism of oxidative stress-induced VSMC apoptosis is still poorly understood. Here, we aimed to analyse the role of soluble adenylyl cylclase (sAC). VSMC derived from rat aorta were treated with either H2O2 (300 µmol/L) or DMNQ (30 µmol/L) for 6 h. Oxidative stress-induced apoptosis was prevented either by treatment with 30 µmol/L KH7 (a specific inhibitor of sAC) or by stable sAC-knockdown (shRNA-transfection). A similar effect was found after inhibition of protein kinase A (PKA). Suppression of the sAC/PKA-axis led to a significant increase in phosphorylation of the p38 mitogen-activated protein kinase under oxidative stress accompanied by a p38-dependent phosphorylation/inactivation of the pro-apoptotic Bcl-2-family protein Bad. Pharmacological inhibition of p38 reversed these effects of sAC knockdown on apoptosis and Bad phosphorylation, suggesting p38 as a link between sAC and apoptosis. Analysis of the protein phosphatases 1 and 2A activities revealed an activation of phosphatase 1, but not phosphatase 2A, under oxidative stress in a sAC/PKA-dependent manner and its role in controlling the p38 phosphorylation. Inhibition of protein phosphatase 1, but not 2A, prevented the pro-apoptotic effect of oxidative stress. In conclusion, sAC/PKA-signaling plays a key role in the oxidative stress-induced apoptosis of VSMC. The cellular mechanism consists of the sAC-promoted and protein phosphatase 1-mediated suppression of p38 phosphorylation resulting to activation of the mitochondrial pathway of apoptosis.

Topics: Adenylyl Cyclases; Animals; Animals, Newborn; Antioxidants; Apoptosis; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Hydrogen Peroxide; Mitochondria; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Naphthoquinones; Oxidants; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Phosphatase 1; Rats; Signal Transduction

Although cystic fibrosis (CF) pathophysiology is explained by a defect in CF transmembrane conductance regulator (CFTR) protein, the broad spectrum of disease severity is the consequence of environmental and genetic factors. Among them, oxidative stress has been demonstrated to play an important role in the evolution of this disease, with susceptibility to oxidative damage, decline of pulmonary function, and impaired lung antioxidant defense. Although oxidative stress has been implicated in the regulation of inflammation, its molecular outcomes in CF cells remain to be evaluated. To address the question, we compared the gene expression profile in NuLi-1 cells with wild-type CFTR and CuFi-1 cells homozygous for ΔF508 mutation cultured at air-liquid interface. We analyzed the transcriptomic response of these cell lines with microarray technology, under basal culture conditions and after 24 h oxidative stress induced by 15 μM 2,3-dimethoxy-1,4-naphtoquinone. In the absence of oxidative conditions, CuFi-1 gene profiling showed typical dysregulated inflammatory responses compared with NuLi-1. In the presence of oxidative conditions, the transcriptome of CuFi-1 cells reflected apoptotic transcript modulation. These results were confirmed in the CFBE41o- and corrCFBE41o- cell lines as well as in primary culture of human CF airway epithelial cells. Altogether, our data point to the influence of oxidative stress on cell survival functions in CF and identify several genes that could be implicated in the inflammation response observed in CF patients.

Topics: Apoptosis; Caspase 3; Caspase 7; Cell Line; Cell Survival; Cells, Cultured; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Down-Regulation; Epithelial Cells; Gene Expression Profiling; Gene Expression Regulation; Gene Ontology; Humans; Inflammation; Lung; Naphthoquinones; Oxidative Stress; Reproducibility of Results; RNA, Messenger; Up-Regulation

Label free time-lapse microscopy has opened a new avenue to the study of time evolving events in living cells. When combined with automated image analysis it provides a powerful tool that enables automated large-scale spatiotemporal quantification at the cell population level. Very few attempts, however, have been reported regarding the design of image analysis algorithms dedicated to the detection of apoptotic cells in such time-lapse microscopy images. In particular, none of the reported attempts is based on sufficiently fast signal processing algorithms to enable large-scale detection of apoptosis within hours/days without access to high-end computers. Here we show that it is indeed possible to successfully detect chemically induced apoptosis by applying a two-dimensional linear matched filter tailored to the detection of objects with the typical features of an apoptotic cell in phase-contrast images. First a set of recorded computational detections of apoptosis was validated by comparison with apoptosis specific caspase activity readouts obtained via a fluorescence based assay. Then a large screen encompassing 2,866 drug like compounds was performed using the human colorectal carcinoma cell line HCT116. In addition to many well known inducers (positive controls) the screening resulted in the detection of two compounds here reported for the first time to induce apoptosis.

Topics: Antibiotics, Antineoplastic; Apoptosis; Caspase 3; Caspase 7; HCT116 Cells; High-Throughput Screening Assays; Humans; Microscopy; Mitomycin; Naphthoquinones; Organic Chemicals; Piperidines; Staining and Labeling; Time-Lapse Imaging

Salvia officinalis (SO) and Thymus vulgaris (TV) are medicinal plants well known for their curative powers. However, the molecular mechanisms responsible for these abilities of sage and thyme have not been fully understood yet. In this study we investigated the composition and the quantitative estimation of plant extracts, the protective effects of plant extracts against hydrogen peroxide- and 2,3-dimethoxy-1,4-naphthoquinone-induced DNA damage, and levels of enzymatic and non-enzymatic antioxidants (superoxide dismutase, glutathione peroxidase, glutathione) in human HepG2 cells. To measure antioxidative activity of plant extracts we used three assays: 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP) and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The results showed that the oxidant-induced DNA lesions were significantly reduced in cells pre-treated with the plant extracts studied. The observed DNA-protective activity could be explained by both elevation of GPx activity in cells pre-treated with SO and TV and antioxidant activity of SO and TV.

Topics: Antioxidants; DNA Damage; Glutathione; Glutathione Peroxidase; Hep G2 Cells; Humans; Hydrogen Peroxide; Naphthoquinones; Oxidants; Plant Extracts; Salvia officinalis; Superoxide Dismutase; Thymus Plant

Nitric oxide production by the endothelium is required for normal vascular homeostasis; however, in conditions of oxidative stress, interactions of nitric oxide with reactive oxygen species (ROS) are thought to underlie endothelial dysfunction. Beyond canonical nitric oxide signaling pathways, nitric oxide production results in the post-translational modification of protein thiols, termed S-nitrosation. The potential interplay between S-nitrosation and ROS remains poorly understood and is the focus of the current study. The effects of the S-nitrosating agent S-nitrosocysteine (CysNO) in combination with redox-cycling agents was examined in bovine aortic endothelial cells (BAEC). CysNO significantly impairs mitochondrial function and depletes the NADH/NAD(+) pool; however, these changes do not result in cell death. When faced with the additional stressor of a redox-cycling agent used to generate ROS, further loss of NAD(+) occurs, and cellular ATP pools are depleted. Cellular S-nitrosothiols also accumulate, and cell death is triggered. These data demonstrate that CysNO sensitizes endothelial cells to redox-cycling agent-dependent mitochondrial dysfunction and cell death and identify attenuated degradation of S-nitrosothiols as one potential mechanism for the enhanced cytotoxicity.

Topics: Animals; Aorta; Cattle; Cell Death; Cells, Cultured; Cysteine; Drug Synergism; Endothelial Cells; Mitochondria; Naphthoquinones; Nitrosation; Reactive Oxygen Species; S-Nitrosothiols

Reactive oxygen species (ROS) are known to mediate a variety of biological responses, including cell motility. Recently, we indicated that lysophosphatidic acid (LPA) receptor-3 (LPA3) increased cell motile activity stimulated by hydrogen peroxide. In the present study, we assessed the role of LPA1 in the cell motile activity mediated by ROS in mouse fibroblast 3T3 cells. 3T3 cells were treated with hydrogen peroxide and 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) at concentrations of 0.1 and 1 μM for 48 h. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3 cells treated with hydrogen peroxide and DMNQ were significantly higher than those of untreated cells. 3T3 cells treated with hydrogen peroxide and DMNQ showed elevated expression levels of the Lpar3 gene, but not the Lpar1 and Lpar2 genes. To investigate the effects of LPA1 on the cell motile activity induced by hydrogen peroxide and DMNQ, Lpar1-overexpressing (3T3-a1) cells were generated from 3T3 cells and treated with hydrogen peroxide and DMNQ. The cell motile activities stimulated by hydrogen peroxide and DMNQ were markedly suppressed in 3T3-a1 cells. These results suggest that LPA signaling via LPA1 inhibits the cell motile activities stimulated by hydrogen peroxide and DMNQ in 3T3 cells.

Topics: 3T3 Cells; Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Diphosphates; Fibroblasts; Gene Expression Regulation; Glycerol; Hydrogen Peroxide; Lysophospholipids; Mice; Naphthoquinones; Neuroblastoma; Rats; Receptors, Lysophosphatidic Acid

Glucocorticoids (GCs) are essential for normal brain development; however, there is consistent evidence that prenatal exposure of the fetal brain to excess GCs permanently modifies the phenotype of neuronal cells. In this paper, the murine-derived multipotent stem cell line C17.2 was used, as an in vitro model, to investigate the impact of GCs on neural stem cell survival. Our results indicate that dexamethasone (Dex) increases the sensitivity of murine neural stem cells (NSCs) to 2,3-methoxy-1,4-naphthoquinone-induced apoptosis, and this effect could be blocked by the glucocorticoid-receptor (GR) antagonist mifepristone, strongly suggesting the involvement of the GR. Furthermore, our results show that Dex decreases cell number and induces a G1-arrest. We hypothesized that the mitochondria are the main target of Dex. Interestingly, after treatment with Dex, 72% of the investigated genes involved in the mitochondrial respiratory chain are down-regulated, as well as 29% of the genes encoding for antioxidant enzymes. In conclusion, using the C17.2 cell line as a model to study developmental neurotoxicity in vitro, we have shown that GCs can increase cellular sensitivity to oxidative stress and alter the phenotype of NCSs.

Topics: Animals; Anti-Inflammatory Agents; Apoptosis; Blotting, Western; Caspase 3; Cell Cycle; Cell Death; Cell Line; Cell Nucleus; Dexamethasone; Electron Transport; Flow Cytometry; G1 Phase; Gene Expression; Mice; Mifepristone; Naphthoquinones; Neural Stem Cells; Oxidative Stress; Real-Time Polymerase Chain Reaction; Receptors, Glucocorticoid; Trypan Blue

Quinone toxicity is induced by two principal mechanisms: arylation/alkylation and a redox cycle. We have previously shown that increases in intracellular levels of superoxide anion and cell death induced by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), a redox cycling quinone, are enhanced by pretreatment of rat primary hepatocytes with cytochrome P450 inhibitors. This indicates a novel interaction of quinones with cytochrome P450, and is thus worthy of further investigation using an in vivo model. The aim of this study was to examine the effects of cytochrome P450 inhibitors on DMNQ-induced hepatotoxicity in rats. When DMNQ was administered intraperitoneally, the activities of serum alanine aminotransferase and aspartate aminotransferase were found to increase in a dose-dependent manner, indicating that hepatotoxicity was induced by treatment with DMNQ. Pretreatment with the cytochrome P450 inhibitors SKF-525A (SKF), cimetidine and ketoconazole potentiated the DMNQ-induced hepatotoxicity. The blood concentration of DMNQ was not affected by administration of SKF. Pretreatment with the antioxidant α-tocopherol almost completely attenuated the hepatotoxicity induced by DMNQ and by the combination of DMNQ with SKF. Levels of reduced glutathione in the liver were decreased and levels of oxidized glutathione were increased by treatment with DMNQ. These effects were potentiated by pretreatment with SKF. DMNQ-induced lipid peroxidation in the liver was also enhanced by pretreatment with SKF. Taken together, these results indicate that DMNQ-induced hepatotoxicity is augmented by inhibition of cytochrome P450 and that this augmentation is due to the enhancement of oxidative stress.

Topics: alpha-Tocopherol; Animals; Antioxidants; Chemical and Drug Induced Liver Injury; Cimetidine; Cytochrome P-450 Enzyme Inhibitors; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Glutathione; Ketoconazole; Lipid Peroxidation; Liver; Male; Naphthoquinones; Oxidation-Reduction; Oxidative Stress; Proadifen; Rats; Rats, Wistar; Substrate Cycling

Alterations in intrauterine programming occurring during critical periods of development have adverse consequences for whole-organ systems or individual tissue functions in later life. In this paper, we show that rat embryonic neural stem cells (NSCs) exposed to the synthetic glucocorticoid dexamethasone (Dex) undergo heritable alterations, possibly through epigenetic mechanisms. Exposure to Dex results in decreased NSC proliferation, with no effects on survival or differentiation, and changes in the expression of genes associated with cellular senescence and mitochondrial functions. Dex upregulates cell cycle-related genes p16 and p21 in a glucocorticoid receptor(GR)-dependent manner. The senescence-associated markers high mobility group (Hmg) A1 and heterochromatin protein 1 (HP1) are also upregulated in Dex-exposed NSCs, whereas Bmi1 (polycomb ring finger oncogene) and mitochondrial genes Nd3 (NADH dehydrogenase 3) and Cytb (cytochrome b) are downregulated. The concomitant decrease in global DNA methylation and DNA methyltransferases (Dnmts) suggests the occurrence of epigenetic changes. All these features are retained in daughter NSCs (never directly exposed to Dex) and are associated with a higher susceptibility to oxidative stress, as shown by the increased occurrence of apoptotic cell death on exposure to the redox-cycling reactive oxygen species (ROS) generator 2,3-dimethoxy-1-naphthoquinone (DMNQ). Our study provides novel evidence for programming effects induced by glucocorticoids (GCs) on NSCs and supports the idea that fetal exposure to endogenous or exogenous GCs is likely to result in long-term consequences that may predispose to neurodevelopmental and/or neurodegenerative disorders.

Topics: Animals; Cell Proliferation; Cellular Senescence; Chromobox Protein Homolog 5; Chromosomal Proteins, Non-Histone; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes b; Dexamethasone; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Epigenesis, Genetic; Glucocorticoids; Mitochondria; NADH Dehydrogenase; Naphthoquinones; Neural Stem Cells; Nuclear Proteins; Polycomb Repressive Complex 1; Proto-Oncogene Proteins; Rats; Receptors, Glucocorticoid; Repressor Proteins

Reactive oxygen species (ROS) display cytotoxicity that can be exacerbated by iron. Paradoxically, HeLa cells treated with the ROS-generators menadione and 2,3-dimethoxy-1,4-naphthoquinone display increased free labile iron. HeLa cells exposed to ROS undergo apoptosis but iron chelation limits the extent of cell death suggesting the rise in intracellular iron plays a signaling role in this pathway. This idea is supported by the fact that iron chelation also alters the pattern of ROS-induced phosphorylation of stress-activated protein kinases SAPK/JNK and p38 MAPK. Thus, ROS-induced increases in cellular free iron contribute to signaling events triggered during oxidative stress response.

Topics: Apoptosis; Blotting, Western; HeLa Cells; Humans; Iron; MAP Kinase Kinase 4; Naphthoquinones; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Signal Transduction; Vitamin K 3; Vitamins

2,3-dimethoxy-1,4-naphthoquinone (CAS-RN 6959-96-3) (DMNQ) and 2-methyl-1,4-naphthoquinone (CAS-RN 58-27-5) (MNQ:menadione) are effective one electron redox cycling chemicals in vitro. In addition, in vitro MNQ forms a thioether conjugate with glutathione by nucleophilic attack at the third carbon. In contrast, here we demonstrate that in vivo the major metabolic route is directly to the dihydronaphthoquinone for both DMNQ and MNQ followed by conjugation to mono- and di-glucuronides and sulfate. Analysis of urine and bile showed that glutathione conjugation of MNQ was only a very minor route of metabolism. DMNQ was distributed to all tissues including the brain, and MNQ was much less widely distributed. For DMNQ tissue half-life, in particular for the heart, was considerably longer than the plasma half-life. For both DMNQ and MNQ, urine 8-oxo-7,8-dihydro-2'-deoxyguanosine and liver transcriptomic analysis failed to show any evidence of redox stress. Oxidized glutathione (GSSG) in liver increased significantly at the 10 min postdosing time point only. Metabonomic analysis 96 h after DMNQ administration indicated decreased liver glucose and increased lactate and creatine suggesting an impairment of oxidative metabolism. We conclude that in vivo DMNQ and MNQ are primarily two electron reduced to the dihydronaphthoquinones and undergo little one electron redox cycling. For DMNQ, disruption of cellular oxidative metabolism may be a primary mechanism of toxicity rather than redox stress.

Topics: Animals; Chromatography, Liquid; Creatinine; Electrons; Liver; Male; Metabolomics; Mice; Mice, Inbred C57BL; Naphthoquinones; Oxidative Stress; Tandem Mass Spectrometry; Tissue Distribution; Transcription, Genetic; Vitamin K 3

The tetraheme c-type cytochrome, CymA, is essential for arsenate respiratory reduction in Shewanella sp. ANA-3, a model arsenate reducer. CymA is predicted to mediate electron transfer from quinols to the arsenate respiratory reductase (ArrAB). Here, we present biochemical and physiological evidence that CymA interacts with menaquinol (MQH(2)) substrates. Fluorescence quench titration with the MQH(2) analog, 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), was used to demonstrate quinol binding of E. coli cytoplasmic membranes enriched with various forms of CymA. Wild-type CymA bound HOQNO with a K (d) of 0.1-1 microM. It was also shown that the redox active MQH(2) analog, 2,3-dimethoxy-1,4-naphthoquinone (DMNH(2)), could reduce CymA in cytoplasmic membrane preparations. Based on a CymA homology model made from the NrfH tetraheme cytochrome structure, it was predicted that Lys91 would be involved in CymA-quinol interactions. CymA with a K91Q substitution showed little interaction with HOQNO. In addition, DMNH(2)-dependent reduction of CymA-K91Q was diminished by 45% compared to wild-type CymA. A DeltacymA ANA-3 strain containing a plasmid copy of cymA-K91Q failed to grow with arsenate as an electron acceptor. These results suggest that Lys91 is physiologically important for arsenate respiration and support the hypothesis that CymA interacts with menaquinol resulting in the reduction of the cytochrome.

Topics: Amino Acid Sequence; Arsenate Reductases; Arsenates; Bacterial Proteins; Cell Membrane; Cytochrome c Group; Electron Transport; Escherichia coli; Hydroxyquinolines; Lysine; Molecular Sequence Data; Mutagenesis, Site-Directed; Naphthoquinones; Oxidation-Reduction; Shewanella

In this study, two commercially available superoxide scavengers, tetrakis (1-methyl-4-pyridyl) porphyrin (Mn[III]TMPyP) and superoxide dismutase (SOD), as well as red palm oil (RPO), a natural vegetable oil, had been used to investigate their possible in vitro effects against the toxic effects of superoxide (O(2).) on human sperm motility. Semen samples were obtained from 12 normozoospermic healthy volunteer donors aged between 19 and 23 years. The O(2). donor 2,3-dimetoxyl-1,4-naphthoquinone (DMNQ) (2.5 micromol L(-1)-100 micromol L(-1)) was added to normozoospermic post-swim-up sperm in the presence or absence of Mn(III)TMPyP (50 micromol L(-1)), SOD (50 IU) or RPO (0.1% or 0.5%). Computer-assisted semen analysis was used to analyze various motility parameters. The parameters of interest were percentage of motile cells, progressive motility, rapid cells and static cells. Concentrations of higher than 25 micromol L(-1) DMNQ were detrimental to sperm motility. Mn(III)TMPyP was able to attenuate the effect of O(2). on the motility parameters. In vitro addition of SOD and RPO showed harmful effects on sperm motility.

Topics: Antioxidants; Humans; Male; Metalloporphyrins; Naphthoquinones; Palm Oil; Plant Oils; Sperm Motility; Superoxides; Young Adult

Skeletal muscles produce transient reactive oxygen species (ROS) in response to intense stimulation, disuse atrophy, heat stress, hypoxia, osmotic stress, stretch and cell receptor activation. The physiological significance is not well understood. Protein phosphatases (PPases) are known to be highly sensitive to oxidants and could contribute to many different signalling responses in muscle. We tested whether broad categories of PPases are inhibited by levels of acute oxidant exposure that do not result in loss of contractile function or gross oxidative stress. We also tested if this exposure results in elevated levels of global protein phosphorylation. Rat diaphragm muscles were treated with either 2,3-dimethoxy-1-naphthoquinone (DMNQ; 1, 10, 100 microm; a mitochondrial O(2)(.-)/H2O2 generator) or exogenous H2O2 (5, 50, 500 microm) for 30 min. Supernatants were assayed for serine/threonine PPase (Ser/Thr-PPase) or protein tyrosine PPase (PTP) activities. With the exception of 500 microm H2O2, no other oxidant exposures significantly elevated protein carbonyl formation, nor did they alter the magnitude of twitch force. DMNQ significantly decreased all categories of PPase activity at 10 and 100 microm and reduced PTP at 1 microm. Similar reductions in Ser/Thr-PPase activity were seen in response to 50 and 500 microm H2O2 and PTP at 500 microm H2O2. ROS treatments resulted a dose-dependent increase in the phosphorylation states of many proteins. The data are consistent with the concept that PPases, within intact skeletal muscles, are highly sensitive to acute changes in ROS activity and that localized ROS play a critical role in lowering the barriers for effective phosphorylation events to occur in muscle cells, thus increasing the probability for cell signalling responses to proceed.

Topics: Animals; Dose-Response Relationship, Drug; Hydrogen Peroxide; In Vitro Techniques; Kinetics; Male; Muscle Contraction; Muscle Proteins; Muscle, Skeletal; Naphthoquinones; Oxidants; Oxidation-Reduction; Oxidative Stress; Phosphoprotein Phosphatases; Phosphorylation; Protein Carbonylation; Protein Tyrosine Phosphatases; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Signal Transduction

Resveratrol, an important antioxidant found in grapes and wine, is likely to contribute to red wine's potential to prevent human cardiovascular disease. In addition to its known (direct) antioxidant effect, we have found that resveratrol also regulates the gene expression of pro-oxidative and anti-oxidative enzymes in human endothelial cells. NADPH oxidases (Nox) are the predominant producers of superoxide in the vasculature, whereas superoxide dismutase (SOD) and glutathione peroxidase 1 (GPx1) are the major enzymes responsible for the inactivation of superoxide and hydrogen peroxide, respectively. Incubation of human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 cells with resveratrol resulted in a concentration- and time-dependent downregulation of Nox4, the most abundant NADPH oxidase catalytic subunit (quantitative real-time RT-PCR). The same resveratrol regimen upregulated the mRNA expression of SOD1 and GPx1. The addition the protein levels of SOD1 and GPx1 were enhanced by resveratrol in a concentration-dependent manner (Western blot analyses). Pretreatment of EA.hy 926 cells with resveratrol completely abolished DMNQ-induced oxidative stress. Thus, the expressional suppression of pro-oxidative genes (such as NADPH oxidase) and induction of anti-oxidative enzymes (such as SOD1 and GPx1) might be an important component of the vascular protective effect of resveratrol.

Topics: Antioxidants; Blotting, Western; Cells, Cultured; Endothelial Cells; Endothelium, Vascular; Gene Expression Regulation, Enzymologic; Glutathione Peroxidase; Glutathione Peroxidase GPX1; Humans; NADPH Oxidase 4; NADPH Oxidases; Naphthoquinones; Oxidative Stress; Reactive Oxygen Species; Resveratrol; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stilbenes; Superoxide Dismutase; Superoxide Dismutase-1

The development of therapeutic strategies to inhibit reactive oxygen species (ROS)-mediated damage in blood vessels has been limited by a lack of specific targets for intervention. Targeting ROS-mediated events in the vessel wall is of interest, because ROS play important roles throughout atherogenesis. In early atherosclerosis, ROS stimulate vascular smooth muscle cell (VSMC) growth, whereas in late stages of lesion development, ROS induce VSMC apoptosis, causing atherosclerotic plaque instability. To identify putative protective genes against oxidative stress, mouse aortic VSMC were infected with a retroviral human heart cDNA expression library, and apoptosis was induced in virus-infected cells by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) treatment. A total of 17 different, complete cDNAs were identified from the DMNQ-resistant VSMC clones by PCR amplification and sequencing. The cDNA encoding PP1cgamma1 (catalytic subunit of protein phosphatase 1) was present in several independent DMNQ-resistant VSMC clones. DMNQ increased mitochondrial ROS production, caspase-3/7 activity, DNA fragmentation, and decreased mitochondrial transmembrane potential in VSMC while decreasing PP1cgamma1 activity and expression. Depletion of PP1cgamma1 expression by short hairpin RNA significantly enhanced basal as well as DMNQ-induced VSMC apoptosis. PP1cgamma1 overexpression abrogated DMNQ-induced JNK1 activity, p53 Ser(15) phosphorylation, and Bax expression and protected VSMC against DMNQ-induced apoptosis. In addition, PP1cgamma1 overexpression attenuated DMNQ-induced caspase-3/7 activation and DNA fragmentation. Inhibition of p53 protein expression using small interfering RNA abrogated DMNQ-induced Bax expression and significantly attenuated VSMC apoptosis. Together, these data indicate that PP1cgamma1 overexpression promotes VSMC survival by interfering with JNK1 and p53 phosphorylation cascades involved in apoptosis.

Topics: Animals; Apoptosis; Drug Resistance; Gene Expression Regulation; Gene Library; Gene Silencing; Heart; Humans; Hydrogen Peroxide; Membrane Potentials; Mice; Mice, Inbred C57BL; Mitochondria; Muscle, Smooth, Vascular; Naphthoquinones; Oxidants; Oxidative Stress; Proteasome Endopeptidase Complex; Protein Phosphatase 1; Reactive Oxygen Species; RNA, Small Interfering; Signal Transduction

Utilizing primary rat hepatocytes we investigated the potential antimutagenic and anti-cytotoxic effects of carboxymethyl chitin-glucan (CM-CG) with respect to oxidative stress induced by the model free-radical-generating compounds hydrogen peroxide (H2O2) or 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). Different kinds of CM-CG action were studied by two different treatment protocols: a. pre-incubation of freshly isolated hepatocytes with the potential anti-mutagen followed by exposure to the oxidant or b. simultaneous treatment of hepatocytes with the potential anti-mutagen and the oxidant.. As a measure of genotoxicity, the percentages of DNA in tails of comets by single cell gel electrophoresis were evaluated. The cytotoxicological endpoints analysed were the cell density (number of cells/cm2), and the percentages of apoptotic and necrotic cells.. H2O2 and DMNQ, causing DNA single-strand breaks via the formation of *OH radicals, have been demostrated to induce both genotoxic and cytotoxic effects in primary rat hepatocytes resulting in increased percentages of DNA in tails of comets, and increased frequencies of apoptotic and necrotic cells accompanied by a decreased cell density. Further investigations were therefore focussed on possible modifications of these parameters by CM-CG. The results obtained clearly demonstrate that CM-CG (applied before and during treatment) protects primary rat hepatocytes against the genotoxic and cytotoxic effects of oxidative stress (H2O2 or DMNQ), whereas CM-CG itself has no effect on the endpoints of genotoxicity and cytotoxicity studied.. Our results indicate that carboxymethyl chitin-glucan represents a natural fungal polysaccharide that can inhibit the genotoxicity and cytotoxicity of experimentally induced oxidative stress in primary rat hepatocytes.

Topics: Animals; Antioxidants; Apoptosis; Cell Count; Cell Death; Cell Separation; Chitin; Comet Assay; DNA; DNA Damage; Female; Glucans; Hepatocytes; Hydrogen Peroxide; Mutagenicity Tests; Mutagens; Naphthoquinones; Necrosis; Oxidants; Protective Agents; Rats; Rats, Inbred F344

Cytotoxicity of quinones has been attributed to free radical generation and to arylation of cellular nucleophiles. For redox-cycling quinones, cell injury is associated with mitochondrial permeability transition, whereas arylating quinones directly depolarise the mitochondrial membrane and deplete ATP. Like mitochondrial electron transport, plasma membrane electron transport (PMET), plays a multifaceted role in cellular redox homeostasis but the effects of quinones on PMET are unknown. Here we investigate the effects of redox-cycling 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), arylating 1,4-benzoquinone (BQ) and mixed mechanism 2-methyl-1,4-naphthoquinone (MNQ) on PMET, viability and growth of P815 mouse mastocytoma cells.BQ and MNQ rapidly and extensively inhibited PMET as determined by WST-1 /mPMS reduction (IC50 3.5-5 microM at 30 min) whereas the effects of DMNQ were less pronounced. In contrast, MTT reduction (cytosolic NADH dehydrogenase activity over 30 min) was weakly inhibited by BQ (IC50 20 microM) but not by MNQ or DMNQ and cell viability was unaffected. Inhibition of WST-1/mPMS reduction by BQ and MNQ but not DMNQ was fully reversed by NAC. Treatment with DMNQ, MNQ and to a lesser extent BQ inhibited cell proliferation as determined by MTT reduction at 48 h. The effects of BQ and MNQ were reversed by NAC through covalent bonding to BQ and MNQ, but not DMNQ. These results show that arylating quinones are more potent inhibitors of PMET than pure redox-cycling quinones, but that redox-cycling quinones are more cytotoxic.

Topics: Animals; Benzoquinones; Cell Line, Tumor; Cell Membrane; Cell Survival; Electron Transport; Mice; Naphthoquinones; Oxidation-Reduction; Quinones; Vitamin K 3

The present study was designed to investigate the effect of nicotine and polyaromatic hydrocarbon compounds on cerebral endothelial cells (CECs). Nicotine treatments from 15 min to 5h did not cause any changes in the expression and localization of principal junctional proteins. One day of treatment with a relatively high concentration of nicotine induced a decrease in the expression of the tight junction protein ZO-1, occludin, and the adherens junction protein, cadherin. Treatment with 3 x 10(-5)M phenanthrene for 24h caused a redistribution of occludin from the Triton X-100 insoluble to the Triton X-100 soluble fraction. Transendothelial electrical resistance was not significantly affected by 24h treatments with nicotine, methylanthracene or phenanthrene. However, 24h nicotine treatment increased transendothelial permeability in CECs exposed to oxidative stress. Both nicotine and phenanthrene were able to regulate the expression of a large number of proteins as revealed by 2D electrophoresis. Our experiments suggest that tobacco smoking may affect the junctional complex of CECs, and that this effect is enhanced by oxidative stress.

Topics: Animals; beta Catenin; Blood-Brain Barrier; Cadherins; Cerebral Cortex; Claudin-5; Coculture Techniques; Endothelial Cells; Membrane Proteins; Molecular Sequence Data; Naphthoquinones; Neuroglia; Nicotine; Nicotinic Agonists; Occludin; Oxidative Stress; Phenanthrenes; Phosphoproteins; Polycyclic Aromatic Hydrocarbons; Proteome; Rats; Rats, Wistar; Zonula Occludens-1 Protein; Zonula Occludens-2 Protein

Uncoupling protein-2 (UCP2) is a member of the inner mitochondrial membrane anion-carrier superfamily. Although mRNA for UCP2 is widely expressed, protein expression is detected in only a few cell types, including macrophages. UCP2 functions by an incompletely defined mechanism, to reduce reactive oxygen species production during mitochondrial electron transport. We observed that the abundance of UCP2 in macrophages increased rapidly in response to treatments (rotenone, antimycin A and diethyldithiocarbamate) that increased mitochondrial superoxide production, but not in response to superoxide produced outside the mitochondria or in response to H2O2. Increased UCP2 protein was not accompanied by increases in ucp2 gene expression or mRNA abundance, but was due to enhanced translational efficiency and possibly stabilization of UCP2 protein in the inner mitochondrial membrane. This was not dependent on mitochondrial membrane potential. These findings extend our understanding of the homeostatic function of UCP2 in regulating mitochondrial reactive oxygen production by identifying a feedback loop that senses mitochondrial reactive oxygen production and increases inner mitochondrial membrane UCP2 abundance and activity. Reactive oxygen species-induction of UCP2 may facilitate survival of macrophages and retention of function in widely variable tissue environments.

Topics: Antimycin A; Ditiocarb; Hydrogen Peroxide; Ion Channels; Macrophages; Membrane Potential, Mitochondrial; Mitochondrial Membranes; Mitochondrial Proteins; Naphthoquinones; Oxidative Stress; Reactive Oxygen Species; RNA, Messenger; Rotenone; Uncoupling Protein 2

Oxidative stress occurs as a consequence of disturbance in the balance between the generation of reactive oxygen species (ROS) and the antioxidant defence mechanisms. The interaction of ROS with DNA can cause single-, or double-strand breaks that subsequently can lead to the activation of p53, which is central for the regulation of cellular response, e.g. apoptosis, to a range of environmental and intracellular stresses. Previous reports have suggested a regulatory role of p53 in the early activation of caspase-2, upstream of mitochondrial apoptotic signaling. Here we show that excessive ROS formation, induced by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) exposure, induces apoptosis in primary cultured neural stem cells (NSCs) from cortices of E15 rat embryos. Following DMNQ exposure cells exhibited apoptotic hallmarks such as Bax oligomerization and activation, cytochrome c release, caspase activation and chromatin condensation. Additionally, we could show early p53 accumulation and a subsequent activation of caspase-2. The attenuation of caspase-2 activity with selective inhibitors could antagonize the mitochondrial signaling pathway and cell death. Overall, our results strongly suggest that DMNQ-induced oxidative stress causes p53 accumulation and consequently caspase-2 activation, which in turn initiates apoptotic cell death via the mitochondria-mediated caspase-dependent pathway in NSCs.

Topics: Animals; Apoptosis; Cysteine Endopeptidases; Embryonic Stem Cells; Enzyme Activation; Naphthoquinones; Neurons; Oxidative Stress; Rats; Tumor Suppressor Protein p53; Up-Regulation

The 27-kDa heat shock protein (Hps27) is phosphorylated in a way that appears to regulate antioxidant defenses by mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2), a component of the p38(MAPK) pathway. To investigate the role of Hsp27 in cellular resistance to oxidant stress, lung cells (A549) were incubated with MAPK inhibitors to investigate the pathway's role in antioxidant defense. Cells were harvested for measurement of reduced gluthathione and glutathione disulfide (GSH and GSSH); or, exposed to 2,3-dimethoxy-1,4-napthoquinone (DMNQ). Inhibition of MAPK with SB203580 decreased total cellular glutathione (mean +/- SE): Vehicle, 150 +/- 20 mu M; SB203580, 57 +/- 10* (*P < .01). Inhibition of MAPK tripled [GSSG]/[GSH]: Vehicle, 0.29 +/- 0.09; SB203580, 1.06 +/- 0.43* (*P > .05; n = 6 per group). Hsp27 protein content did not change significantly after MAPK inhibition: Vehicle 2.20 +/- 0.24 ng/mg protein; SB203580, 2.03 +/- 0.34 (P > .05). Transfection of epithelial cells with wild-type (pcDNA-HA-Hsp27) or phosphomimic (pcDNA-HA-Hsp27-S3D) vector increased Hsp27 protein, which significantly protected cells from oxidant stress. Inhibition of the MAPK system, including p38(MAPK), results in cellular oxidant stress. Hsp27, which is phosphorylated by MK2 in the MAPK pathway, protects epithelial cells from oxidant stress.

Topics: Adenocarcinoma, Papillary; Cell Line, Tumor; Enzyme Inhibitors; Epithelial Cells; Glutathione; Glutathione Disulfide; Humans; Imidazoles; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Naphthoquinones; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Pyridines; Respiratory Mucosa; Transfection

Microarray technology is proving to be a useful tool to classify undefined environmental toxicants, to investigate underlying mechanisms of toxicity, and to identify candidate toxicant-specific genetic markers by examining global effects of putative toxicants on gene expression profiles. The aim of this study was to evaluate the toxicities of six heavy metals through the comparison with gene expression patterns induced by well-known chemicals. For this purpose, we first identified the genes altered specifically in HepG2 under the exposure of 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), phenol, and N-nitrosodimethylamine (DMN), which were selected as the model chemicals, using DNA microarray. On the basis of the expression profiles of these genes, toxicities of six heavy metals, arsenic, cadmium, nickel, antimony, mercury, and chromium, were evaluated. The specific gene alteration and hierarchical clustering revealed that biological action of six heavy metals was clearly related to that of DMNQ which has been reported to be a reactive oxygen species (ROS) generating chemical and which induced the genes associated with cell proliferative responses. These results suggest that cell proliferative responses which are probably caused by ROS are a major apparent biological action of high-dose heavy metals, supporting the previous reports. Overall, a mechanism-based classification by DNA microarray would be an efficient method for evaluation of toxicities of environmental samples.

Topics: Cell Line, Tumor; Cluster Analysis; Gene Expression Profiling; Gene Expression Regulation; Humans; Metals, Heavy; Naphthoquinones; Oligonucleotide Array Sequence Analysis; Phenol; Principal Component Analysis

Since it has to be expected that individuals exposed to oxidative stress who take supplements of beta-carotene are simultaneously exposed to both beta-carotene cleavage products (CPs) and oxidative stress, and both exposures have been demonstrated to cause genotoxic effects in primary rat hepatocytes, cyto- and genotoxic effects on primary rat hepatocytes after supplementation of the medium with increasing concentrations of a CP mixture during exposure to oxidative stress by treatment with either DMNQ (2,3-dimethoxy-1,4-naphthoquinone) or hypoxia/reoxygenation (Hy/Reox) was investigated. The cytological endpoints analysed were the mitotic indices, the percentages of apoptotic and necrotic cells, the percentages of micronucleated (MN) cells and the number of chromosomal aberrations (CAs) and sister chromatid exchanges (SCE). The results obtained clearly demonstrate that the CP mixture enhances the genotoxic effects of oxidative stress exposure, whereas it had no effect at all on the endpoints of cytotoxicity studied. These results further support the hypothesis that CP might be responsible for the reported carcinogenic response in the beta-CArotene and Retinol Efficacy Trial (CARET) and Alpha-Tocopherol Beta-carotene Cancer prevention (ATBC) chemoprevention trials.

Topics: Animals; beta Carotene; Chromosome Aberrations; DNA Damage; Dose-Response Relationship, Drug; Female; Hepatocytes; Hypoxia; Metaphase; Naphthoquinones; Oxidative Stress; Oxygen; Rats; Rats, Inbred F344

Two mechanisms have been proposed to explain quinone cytotoxicity: oxidative stress via the redox cycle and the arylation of intracellular nucleophiles. As the redox cycle is catalyzed by NADPH cytochrome P450 reductase, cytochrome P450 systems are expected to be related to the cytotoxicity induced by redox-cycling quinones. Thus, we investigated the relationship between cytochrome P450 systems and quinone toxicity for rat primary hepatocytes using an arylator, 1,4-benzoquinone (BQ), and a redox cycler, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). The hepatocyte toxicity of both BQ and DMNQ increased in a time- and dose-dependent manner. Pretreatment with cytochrome P450 inhibitors, such as SKF-525A (SKF), ketoconazole and 2-methy-1,2-di-3-pyridyl-1-propanone, enhanced the hepatocyte toxicity induced by DMNQ but did not affect BQ-induced hepatocyte toxicity. The production of superoxide anion and the levels of glutathione disulfide and thiobarbituric-acid-reactive substances were increased by treatment with DMNQ, and SKF pretreatment further enhanced their increases. In addition, NADPH oxidation in microsomes was increased by treatment with DMNQ and further augmented by pretreatment with SKF, and a NADPH cytochrome P450 reductase inhibitor, diphenyleneiodonium chloride completely suppressed NADPH oxidations increased by treatment with either DMNQ- or DMNQ + SKF. Pretreatment with antioxidants, such as alpha-tocopherol, reduced glutathione, N-acetyl cysteine or an iron ion chelator deferoxamine, totally suppressed DMNQ- and DMNQ + SKF-induced hepatocyte toxicity. These results indicate that the hepatocyte toxicity of redox-cycling quinones is enhanced under cytochrome P450 inhibition, and that this enhancement is caused by the potentiation of oxidative stress.

Topics: Animals; Antioxidants; Benzoquinones; Catecholamines; Cell Survival; Cytochrome P-450 Enzyme Inhibitors; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Inhibitors; Glutathione Disulfide; Hepatocytes; Imidazolines; Iron Chelating Agents; Ketoconazole; L-Lactate Dehydrogenase; Lipid Peroxidation; Male; Metyrapone; Molecular Structure; NADP; Naphthoquinones; Oxidative Stress; Proadifen; Rats; Rats, Wistar; Superoxides; Thiobarbituric Acid Reactive Substances

We have shown that Ca2+-mediated protein kinase C (PKC) activation induces impairment of bile salt secretory function and F-actin redistribution in hepatocyte couplets. Because oxidative stress induces Ca2+ elevation, we tested here whether PKC inhibition or protein kinase A (PKA) activation, which often counteracts PKC-dependent effects, can prevent and reverse these alterations. The pro-oxidant compounds tert-butylhydroperoxide (tBOOH, 100 microM) and 2,3-dimethoxy-1,4-naphthoquinone (30 microM), reduced by -41% and -29%, respectively, the percentage of couplets accumulating the fluorescent bile salt analog, cholyl-lysylfluorescein in their canalicular vacuoles (p < 0.01). tBOOH-induced bile salt secretory failure was accompanied by internalization of the canalicular bile salt export pump (Bsep), and disarrangement of cytoskeletal F-actin. All these deleterious effects were fully prevented by the intracellular Ca2+ chelator BAPTA/AM (20 microM), the pan-specific PKC inhibitors H7 (100 microM) and staurosporine (1 microM), the inhibitor of Ca2+-dependent PKCs, Gö6976 (2 microM), and the PKA activator dibutyryl-cAMP (500 microM). H7, Gö6976, and dibutyryl-cAMP not only prevented but also fully reversed the decrease in the cholyl-lysyl-fluorescein accumulation. In conclusion, these results suggest that low levels of oxidative stress impair bile salt secretion by internalizing Bsep through a Ca2+-dependent, PKC-mediated mechanism, and that inhibition of PKC, or activation of PKA, prevents and reverses these effects. Alterations in actin organization may be a causal factor.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B, Member 11; ATP-Binding Cassette Transporters; Bile Acids and Salts; Bucladesine; Calcium; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Hepatocytes; Male; Naphthoquinones; Oxidative Stress; Protein Kinase C; Rats; Rats, Wistar; tert-Butylhydroperoxide

These studies were conducted to determine the effects of oxidative stress on human T cell differentiation and polarization into Th1 or Th2 phenotypes. Highly purified naive CD4+ T cells were isolated from PBMC of healthy, nonatopic donors. CD4+ T cells were stimulated with anti-CD3 and anti-CD28 mAb in the presence or absence of oxidative stress as supplied by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), which generates a low level of superoxide anion. Increases in cellular superoxide were observed by exposure to DMNQ. Exposure of unpolarized CD4+ T cells to IL-12 or IL-4 resulted in a Th1 or Th2 phenotype, respectively. T cells stimulated in the absence of polarizing cytokines secreted modest amounts of IFN-gamma and TNF-alpha. Cells stimulated in the continuous presence of 5 microM DMNQ, displayed a marked up-regulation in Th2 cytokines, including IL-4, IL-5, and IL-13, but not the Th1 cytokine IFN-gamma. Th2 responses were blunted by concomitant exposure to thiol antioxidants. Long-term exposure of T cells to DMNQ resulted in growth of cells expressing CCR4, and a decrease in cells expressing CXCR3, indicating phenotypic conversion to Th2 cells. These results suggest that oxidative stress favors a Th2-polarizing condition.

Topics: Cell Differentiation; Cells, Cultured; Cytokines; Humans; Immunophenotyping; Jurkat Cells; Naphthoquinones; Oxidative Stress; Phosphorylation; Receptors, Chemokine; STAT6 Transcription Factor; Th2 Cells

1,4-Naphthoquinones are widely distributed in nature and many clinically important antitumor drugs containing a quinone moiety, such as anthracyclines, mitoxantrones and saintopin, show excellent anticancer activity. In this study, 2- or 6-substituted 5,8-dimethoxy-1,4-naphthoquinone (DMNQ) and 5,8-dihydroxy-1,4-naphthoquinone (DHNQ) derivatives were synthesized, and their cytotoxic activity against L1210 and P388 cancer cells was examined. Their antitumor activity was also assessed in mice bearing S-180 cells in the peritoneal cavity. In comparison with the DMNQ derivatives, the DHNQ derivatives exhibited more potent bioactivities than the DMNQ derivatives against both L1210 and P388 cells in vitro and S-180 cells in vivo. The ED50 values of the DHNQ derivatives against P388 cells were in the range of 0.18-1.81 microg/mL whereas those of the DMNQ derivatives were in the range of 0.26-40.41 microg/mL. The T/C (%) values of the DHNQ derivatives, 8, 17, 18, 19, and 20, were found to be comparable to or even better than that of adriamycin. It was also observed that the 2-substituted derivatives (8, 19, 20) showed better antitumor activity than the 6-substituted derivatives (7, 17, 18) in the mice bearing S-180 cells in the peritoneal cavity.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Male; Mice; Mice, Inbred ICR; Naphthoquinones; Neoplasm Transplantation; Sarcoma 180; Structure-Activity Relationship

Tissue hypoxia/ischemia are major pathophysiological determinants. Conditions of decreased oxygen availability provoke accumulation and activation of hypoxia-inducible factor-1 (HIF-1). Recent reports demonstrate a crucial role of HIF-1 for inflammatory events. Regulation of hypoxic responses by the inflammatory mediators nitric oxide (NO) and reactive oxygen species (ROS) is believed to be of pathophysiolgical relevance. It is reported that hypoxic stabilization of HIF-1alpha can be antagonized by NO due to its ability to attenuate mitochondrial electron transport. Likely, the formation of ROS could contribute to this effect. As conflicting results emerged from several studies showing either decreased or increased ROS production during hypoxia, we used experiments mimicking hypoxic intracellular ROS changes by using the redox cycling agent 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), which generates superoxide inside cells. Treatment of A549, HEK293, HepG2, and COS cells with DMNQ resulted in a concentration-dependent raise in ROS which correlated with HIF-1alpha accumulation. By using a HIF-1alpha-von Hippel-Lindau tumor suppressor protein binding assay, we show that ROS produced by DMNQ impaired prolyl hydroxylase activity. When HIF-1alpha is stabilized by NO, low concentrations of DMNQ (<1 microM) revealed no effect, intermediate concentrations of 1 to 40 microM DMNQ attenuated HIF-1alpha accumulation and higher concentrations of DMNQ promoted HIF-1alpha stability. Attenuation of NO-induced HIF-1alpha stability regulation by ROS was mediated by an active proteasomal degradation pathway. In conclusion, we propose that scavenging of NO by ROS and vice versa attenuate HIF-1alpha accumulation in a concentration-dependent manner. This is important to fully elucidate HIF-1alpha regulation under inflammatory conditions.

Topics: Acetylcysteine; Active Transport, Cell Nucleus; Apoptosis; Catalase; Cell Extracts; Cell Line; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Naphthoquinones; Nitric Oxide; Peroxynitrous Acid; Protein Biosynthesis; Reactive Oxygen Species; Time Factors; Transcriptional Activation

Evidence is accumulating that capillary endothelial cells (cEC) and astrocytes play a pivotal role in neuroprotection, in particular with respect to counteract oxidative injury. Furthermore, differences among both cell types in response to oxidative stress have been shown and astrocytes seem to be more tolerant in terms of cytotoxicity, however, no reports exist on oxidative stress mediated genotoxicity in astrocytes. We investigated genotoxic and cytotoxic effects of oxidative stress in astrocytes and cECs induced by hypoxia/reoxygenation or by the redox cycling quinone DMNQ. Additionally, the dependence of these effects on glucose availabilty was also studied. On exposure to Hy/Re or 10 muM DMNQ for 24 hr, the frequency of micronucleated and apoptotic cells was significantly increasing, however, astrocytes proved to be more resistant to apoptosis induction, in particular on use of DMNQ. In astrocytes, the low background rates of necrotic cells were not affected and a significant necrosis induction was only detectable in cECs exposed to DMNQ for 24 hr. Short-term exposure to DMNQ (1 hr) had no effect in astrocytes but exerted significant geno- and cytotoxicity in cECs. Increasing the glucose concentration markedly reduced oxidative stress mediated geno- and cytotoxicity in astrocytes. Surprisingly, glucose deprivation (aglycemia) suppressed DMNQ induced micronucleus formation in astrocytes without affecting the frequency of apoptotic cells. Our results indicate that astrocytes are more resistant to oxidative stress than cECs, in particular regarding the potential to counteract genotoxicity as well as apoptosis induction mediated by a short term oxidative insult.

Topics: Analysis of Variance; Animals; Animals, Newborn; Apoptosis; Astrocytes; Cell Death; Cells, Cultured; Cerebral Cortex; Endothelial Cells; Glucose; Mice; Naphthoquinones; Necrosis; Oxidative Stress; Time Factors

(1) There is increasing evidence that the cerebral endothelium and the blood-brain barrier (BBB) plays an important role in the oxidative stress-induced brain damage. The aim of the present study was to investigate the role of interendothelial junctional proteins in the BBB permeability increase induced by oxidative stress. (2) For the experiments, we have used cultured cerebral endothelial cells exposed to hypoxia/reoxygenation or treated with the redox cycling quinone 2,3-Dimethoxy-1,4-naphthoquinone (DMNQ) in the presence or absence of glucose. The expression of junctional proteins and activation of mitogen activated protein kinases (MAPK) was followed by Western-blotting, the interaction of junctional proteins was investigated using coimmunoprecipitation. (3) Oxidative stress induces a downregulation of the tight junction protein occludin expression which is more pronounced in the absence of glucose. Furthermore, oxidative stress leads to disruption of the cadherin-beta-catenin complex and an activation of extracellular signal-regulated kinase (ERK1/2), which is more intense in the absence of glucose. (4) We have shown that one of the causes of the BBB breakdown is probably the structural alteration of the junctional complex caused by oxidative stress, a process in which ERK1/2 may play an important role.

Topics: Animals; beta Catenin; Blood-Brain Barrier; Cadherins; Capillary Permeability; Cell Survival; Cells, Cultured; Cytoskeletal Proteins; Electric Impedance; Endothelial Cells; Extracellular Signal-Regulated MAP Kinases; Glucose; MAP Kinase Signaling System; Membrane Proteins; Mice; Naphthoquinones; Occludin; Oxidative Stress; Tight Junctions; Trans-Activators

Superoxide emerges as key regulatory molecule in many aspects of vascular physiology and disease, but identification of superoxide targets in the vasculature remains elusive. In this work, we investigated the possibility of inhibition of protein phosphatase calcineurin by superoxide in endothelial cells. We employed a redox cycler 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) to generate superoxide inside the cells. DMNQ caused inhibition of cellular calcineurin phosphatase activity, which was reversible upon DMNQ removal. Inhibition was suppressed by pre-incubating the cells with copper/zinc superoxide dismutase (Cu,ZnSOD). In addition, reducing cellular Cu,ZnSOD activity by diethylthiocarbamic acid treatment resulted in calcineurin inhibition and enhanced sensitivity to DMNQ. Further, we could show that DMNQ inhibits calcineurin-dependent nuclear translocation and transcriptional activation of NFAT transcription factor, and Cu,ZnSOD or superoxide scavenger Tiron reduced the inhibition. Thus, superoxide generation in endothelial cells results in inhibition of calcineurin signaling, which could have important pathophysiological implications in the vasculature.

Topics: Animals; Calcineurin; Cattle; Cells, Cultured; Dose-Response Relationship, Drug; Endothelial Cells; Humans; Naphthoquinones; Signal Transduction; Superoxide Dismutase; Superoxides

The activity of hypoxia-inducible factor 1 (HIF-1) is primarily determined by stability regulation of its alpha subunit, which is stabilized under hypoxia but degraded during normoxia. Hydroxylation of HIF-1alpha by prolyl hydroxylases (PHDs) recruits the von Hippel-Lindau (pVHL) E3 ubiquitin ligase complex to initiate proteolytic destruction of the alpha subunit. Hypoxic stabilization of HIF-1alpha has been reported to be antagonized by nitric oxide (NO). By using a HIF-1alpha-pVHL binding assay, we show that NO released from DETA-NO restored prolyl hydroxylase activity under hypoxia. Destabilization of HIF-1alpha by DETA-NO was reversed by free radical scavengers such as NAC and Tiron, thus pointing to the involvement of reactive oxygen species (ROS). Therefore, we examined the effects of ROS on HIF-1alpha stabilization. Treatment of cells under hypoxia with low concentrations of the superoxide generator 2,3-dimethoxy-1,4-naphthoquinone lowered HIF-1alpha protein stabilization. In vitro HIF-1alpha-pVHL interaction assays demonstrated that low-level ROS formation increased prolyl hydroxylase activity, an effect antagonized by ROS scavengers. While determining intracellular ROS formation we noticed that reduced ROS production under hypoxia was restored by the addition of DETA-NO. We propose that an increase in ROS formation contributes to HIF-1alpha destabilization by NO donors under hypoxia via modulation of PHD activity.

Topics: 1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; Acetylcysteine; Carcinoma, Hepatocellular; Cell Line; Humans; Hydroxylation; Hypoxia; Naphthoquinones; Nitric Oxide; Nitric Oxide Donors; Procollagen-Proline Dioxygenase; Reactive Oxygen Species; Triazenes; Tumor Cells, Cultured; Von Hippel-Lindau Tumor Suppressor Protein

Reconciliation of apparently contradictory experimental results obtained on the quinol:fumarate reductase, a diheme-containing respiratory membrane protein complex from Wolinella succinogenes, was previously obtained by the proposal of the so-called "E pathway hypothesis." According to this hypothesis, transmembrane electron transfer via the heme groups is strictly coupled to cotransfer of protons via a transiently established pathway thought to contain the side chain of residue Glu-C180 as the most prominent component. Here we demonstrate that, after replacement of Glu-C180 with Gln or Ile by site-directed mutagenesis, the resulting mutants are unable to grow on fumarate, and the membrane-bound variant enzymes lack quinol oxidation activity. Upon solubilization, however, the purified enzymes display approximately 1/10 of the specific quinol oxidation activity of the wild-type enzyme and unchanged quinol Michaelis constants, K(m). The refined x-ray crystal structures at 2.19 A and 2.76 A resolution, respectively, rule out major structural changes to account for these experimental observations. Changes in the oxidation-reduction heme midpoint potential allow the conclusion that deprotonation of Glu-C180 in the wild-type enzyme facilitates the reoxidation of the reduced high-potential heme. Comparison of solvent isotope effects indicates that a rate-limiting proton transfer step in the wild-type enzyme is lost in the Glu-C180 --> Gln variant. The results provide experimental evidence for the validity of the E pathway hypothesis and for a crucial functional role of Glu-C180.

Topics: Cell Membrane; Crystallography, X-Ray; Electrochemistry; Electron Transport; Electrons; Glutamic Acid; Hydrogen-Ion Concentration; Intracellular Membranes; Kinetics; Membrane Potentials; Models, Biological; Models, Chemical; Models, Molecular; Models, Statistical; Models, Theoretical; Molecular Conformation; Naphthoquinones; Oxidation-Reduction; Oxidoreductases; Oxygen; Protein Conformation; Protons; Solvents; Spectrophotometry; Wolinella

Glutamate cysteine ligase (GCL), composed of a catalytic (GCLC) and modulatory (GCLM) subunit, catalyzes the first step of glutathione (GSH) biosynthesis. Using 4-hydroxy-2-nonenal (4HNE), 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), and tertiary-butylhydroquinone (tBHQ) as models of oxidative stress which are known to work through different mechanisms, we measured changes in cellular GSH, GCL mRNA, and GCL protein. 4HNE and tBHQ treatments increased cellular GSH levels, while DMNQ exposure depleted GSH. Furthermore, changes in the two GCL mRNAs largely paralleled changes in the GCL proteins; however, the magnitudes differed, suggesting some form of translational control. The molar ratio of GCLC:GCLM ranged from 3:1 to 17:1 in control human bronchial epithelial (HBE1) cells and all treatments further increased this ratio. Data from several mouse tissues show molar ratios of GCLC:GCLM that range from 1:1 to 10:1 in support of these findings. These data demonstrate that alterations in cellular GSH are clearly correlated with GCLC to a greater extent than GCLM. Surprisingly, both control HBE1 cells and some mouse tissues have more GCLC than GCLM and GCLM increases to a much lesser extent than GCLC, suggesting that the regulatory role of GCLM is minimal under physiologically relevant conditions of oxidative stress.

Topics: Aldehydes; Antioxidants; Bronchi; Epithelium; Glutamate-Cysteine Ligase; Glutathione; Humans; Hydroquinones; Naphthoquinones; Oxidative Stress

Double staining flow cytometry was performed using 7-amino actinomycin D and 6-carboxy-2', 7'-dichlorodihydrofluorescein diacetate, to detect the level fluctuation of reactive oxygen species (ROS) during the cell cycle of normal NB4 cells. Our results showed that NB4 cells possessed higher level of ROS in G2/M phase than in G1 and S phases. Double staining flow cytometry, with TdT mediated dUTP nick end labeling (Tunel) and propidium iodide (PI), indicated that As2O3 (2 microM) could induce apoptosis in NB4 cells prevailingly from G2/M phase, and this efficacy was enhanced upon co-administration of 2, 3-dimethoxy-1, 4-naphthoquinone (DMNQ) (2.5 microM) which could produce the endogenous ROS. These results suggested that different ROS level in different cell cycle phases of NB4 cells might determine the selective induction of G2/M apoptosis and the cells' susceptibility to apoptosis by As2O3.

Topics: Acetylcysteine; Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Catalase; Cell Cycle; Cell Division; Cell Line, Tumor; Dactinomycin; Flow Cytometry; Fluoresceins; Humans; In Situ Nick-End Labeling; Interphase; Naphthoquinones; Oxides; Propidium; Reactive Oxygen Species

Although the primary function of myoglobin (Mb) has been considered to be cellular O2 storage and supply, recent studies have shown that Mb in addition can act as NO oxidase. Here we report that Mb also significantly contributes to the attenuation of oxidative stress in cardiac muscle. In support of this hypothesis, we found that in isolated perfused hearts of Mb-deficient (myo-/-) mice oxidative challenge by intracoronary infused H2O2 (1-300 microM) or superoxide formed by 2,3-dimethoxy-1,4-naphtoquinone (0.1-30 microM), respectively, depressed cardiac contractility to a greater extent than in wild-type (WT) hearts, e.g., up to [H2O2] = 10 microM there was a significant left ventricular developed pressure (LVDP) decrease in myo-/- hearts only (90.4+/-4.2 vs. 98.1+/-0.7% of control, n=6, P<0.05). Likewise in an ischemia/reperfusion protocol, myo-/- hearts showed a delayed recovery of postischemic function as compared with WT controls (e.g., LVDP was 35.6+/-7.5 vs. 22.4+/-5.3 mmHg, respectively, after 10 min of reperfusion, P<0.05, n=8), which correlated well with an enhanced release of reactive oxygen species in myo-/- hearts as measured by online lucigenin-enhanced chemiluminescence [e.g. 465+/-87 relative light units (RLU) in myo-/- vs. 287+/-73 RLU in WT after 2.5 min of reperfusion, P<0.05, n=8]. (31)P NMR spectroscopy revealed concomitantly a more pronounced phosphocreatine overshoot during reperfusion in the knockout but only minute alterations in ATP and pHi. Our data show that lack of Mb leads to increased vulnerability of cardiac function to oxidative challenge either pharmacologically induced or endogenously generated. We propose that Mb is a key element influencing redox pathways in cardiac muscle to functionally and metabolically protect the heart from oxidative damage.

Topics: Adenosine Triphosphate; Animals; Free Radical Scavengers; Heart Ventricles; Hydrogen Peroxide; Hydrogen-Ion Concentration; Mice; Mice, Knockout; Myocardial Contraction; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Myoglobin; Naphthoquinones; Nuclear Magnetic Resonance, Biomolecular; Oxidation-Reduction; Oxidative Stress; Perfusion; Reactive Oxygen Species; Superoxides

Despite an increasing interest in neural stem cell (NSC) research, relatively little is known about the biochemical regulation of cell death pathways in these cells. We demonstrate here, using murine-derived multipotent C17.2 NSCs, that cells undergo mitochondria-mediated cell death in response to apoptotic stimuli such as oxidative stress induced by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). In particular, treated cells exhibited apoptotic features, including Bax translocation, cytochrome c release, activation of caspase-9 and -3, chromatin condensation and DNA fragmentation. Although C17.2 cells possess the Fas receptor and express procaspase-8, agonistic Fas mAb treatment failed to induce apoptosis. Fas treatment activated the extracellular signal-regulated protein kinase (ERK) pathway, which may have an antiapoptotic as well as a growth stimulating role. Combined, our findings indicate that while NSCs are sensitive to cytotoxic stimuli that involve an engagement of mitochondria, Fas treatment does not induce death and may have an alternative role.

Topics: Amino Acid Chloromethyl Ketones; Animals; Annexin A5; Antibodies, Monoclonal; Benzimidazoles; Blotting, Western; Caspase Inhibitors; Caspases; Cell Death; Cell Differentiation; Cell Line; Chromatin; Coumarins; Cytochromes c; Drug Interactions; Electrophoresis, Gel, Pulsed-Field; Enzyme Inhibitors; fas Receptor; Flavonoids; Fluorescent Dyes; Free Radical Scavengers; Immunohistochemistry; Metalloporphyrins; Mice; Mitochondria; Mitogen-Activated Protein Kinases; Naphthoquinones; Neurons; Oligopeptides; Propidium; Staurosporine; Stem Cells; Subcellular Fractions; Time Factors

Dysregulated cell growth can be caused by increased activity of protein synthesis eukaryotic initiation factor (eIF) 4E. Dysregulated cell growth is also characteristic of atherosclerosis. It is postulated that exposure of vascular cells, such as endothelial cells, smooth muscle cells and monocytes/macrophages, to oxidants, such as oxidized low-density lipoprotein (oxLDL), leads to the elaboration of growth factors and cytokines, which in turn results in smooth muscle cell hyperproliferation. To investigate whether activation of eIF4E might play a role in this hyperproliferative response, vascular cells were treated with oxLDL, oxidized lipid components of oxLDL and several model oxidants, including H(2)O(2) and dimethyl naphthoquinone. Exposure to each of these compounds led to a dose- and time-dependent increase in eIF4E phosphorylation in all three types of vascular cells, correlated with a modest increase in overall translation rate. No changes in eIF4EBP, eIF2 or eIF4B modification state were observed. Increased eIF4E phosphorylation was paralleled by increased presence of eIF4E in high-molecular-mass protein complexes characteristic of its most active form. Anti-oxidants at concentrations typically employed to block oxidant-induced cell signalling likewise promoted eIF4E phosphorylation. The results of this study indicate that increased eIF4E activity may contribute to the pathophysiological events in early atherogenesis by increasing the expression of translationally inefficient mRNAs encoding growth-promoting proteins.

Topics: Animals; Antioxidants; Cells, Cultured; DNA; Endothelium, Vascular; Eukaryotic Initiation Factor-4E; Humans; Hydrogen Peroxide; Linoleic Acids; Lipid Peroxides; Lipoproteins, LDL; Macromolecular Substances; Macrophages; Myocytes, Smooth Muscle; Naphthoquinones; Oxidants; Oxidative Stress; Phosphorylation; Protein Biosynthesis; Rabbits; Reactive Oxygen Species

Cytotoxicity of 1,4-naphthoquinones has been attributed to intracellular reactive oxygen species (ROS) generation through one-electron-reductase-mediated redox cycling and to arylation of cellular nucleophiles. Here, however, we report that in a subclone of lung epithelial A549 cells (A549-S previously called A549-G4S (Watanabe, et al., Am. J. Physiol. 283 (2002) L726-736), the mechanism of ROS generation by menadione and by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), and therefore that of cytotoxicity, differs from the paradigm. Ninety percent of H(2)O(2) generation by both the quinones can be prevented by dicumarol, an inhibitor of NAD(P)H quinone oxidoreductase (NQO1), at the submicromolar level, regardless of the quinone concentrations. Exogenous SOD also inhibits H(2)O(2) production at low but not high concentrations of the quinones, especially DMNQ. Thus, at low quinone concentrations, superoxide-driven hydroquinone autoxidation accounts for more than half of H(2)O(2) generation by both quinones, whereas at high quinone concentrations, especially for DMNQ, comproportionation-driven hydroquinone autoxidation becomes the predominant mechanism. Hydroquinone autoxidation appears to occur predominantly in the extracellular environment than in the cytosol as extracellular catalase can dramatically attenuate quinone-induced cytotoxicity throughout the range of quinone concentrations, whereas complete inactivation of endogenous catalase or complete depletion of intracellular glutathione has only a marginal effect on their cytotoxicity. Finally, we show evidence that ROS production is a consequence of the compensatory defensive role of NQO1 against quinone arylation.

Topics: Buthionine Sulfoximine; Cell Survival; Clone Cells; Humans; Hydrogen Peroxide; Hydroquinones; Lung; Naphthoquinones; Oxidation-Reduction; Reactive Oxygen Species; Respiratory Mucosa; Vitamin K 3

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.

Topics: Adenine; Animals; Benzoquinones; Cell Death; Female; Glutathione; Glutathione Disulfide; Hepatocytes; Male; Molecular Structure; Naphthoquinones; Oncorhynchus mykiss; Oxidation-Reduction; Oxygen; Pyridines; Quantitative Structure-Activity Relationship; Reactive Oxygen Species; Sulfhydryl Compounds; Vitamin K 3

The stress-activated protein kinases SAPK/JNK and p38/mHOG are activated by diverse classes of stress stimuli, many of which induce redox perturbations. We studied the effects of reactive quinones on stress signaling pathways. Menadione (2-methyl-1,4-naphthoquinone), which undergoes both one- and two-electron reduction, completely inhibited SAPK activity at high concentrations while activating SAPK at lower concentrations. Menadione activated p38/mHOG dose responsively. 2,3-Dimethyl-1,4-naphthoquinone (DMNQ), which preferentially undergoes two-electron reduction, had similar effects. In contrast, 1,4-naphthoquinone, which preferentially undergoes one-electron reduction, inhibited SAPK at high concentrations, but failed to activate SAPK at any concentration tested. In addition, this quinone activated p38 only at lower concentrations; high concentrations inhibited p38 activity. These activity profiles correlated with the activation state of the upstream kinase, indicating that the effects were mediated by an upstream step in the kinase pathway. The quinone reductase inhibitor dicoumarol blocked activation of SAPK by menadione and DMNQ, suggesting that two-electron reduction is important. Finally, addition of increasing amounts of hydrogen peroxide mimicked the effects of menadione and DMNQ, suggesting that hydrogen peroxide may be the relevant mediator. Differential activation of stress kinases by reactive quinones demonstrates that the cellular redox environment independently modulates these pathways.

Topics: Animals; Antifibrinolytic Agents; Dicumarol; Dose-Response Relationship, Drug; Electrons; Enzyme Activation; Enzyme Inhibitors; Hydrogen Peroxide; Mice; Mitogen-Activated Protein Kinases; Naphthoquinones; NIH 3T3 Cells; Osmosis; Oxidation-Reduction; p38 Mitogen-Activated Protein Kinases; Protein Isoforms; Quinones; Uncoupling Agents; Vitamin K 3

There is evidence accumulating that brain microvasculature is involved critically in oxidative stress-mediated brain damage. Cultured cerebral microvascular endothelial cells were used to demonstrate the cytotoxic and genotoxic effects elicited by hypoxia/reoxygenation and DMNQ treatment in vitro. In addition, the effect of glucose deprivation during oxidative insult was assessed. The parameters determined were: 1) chromosomal aberrations; 2) induction of micronuclei; and 3) apoptosis. Our results indicate that both the exposure of the cerebral endothelial cells to 24 hr of hypoxia followed by 4 hr of reoxygenation, and treatment with the redox cycling quinone DMNQ, increased markedly the occurrence of chromosomal aberrations and micronuclei. It was found that expression of p53 was induced by oxidative stress, particularly when glucose had been omitted from the culture medium. Aglycemic culture conditions in general exacerbated the cytotoxic effects of oxidative insults, as evidenced by the increase in apoptotic cells and the decrease in the mitotic index. Interestingly, neither an elevation of cell lysis nor an increase in necrosis has been observed during our experiments. In summary, our data indicate that oxidative stress exerts considerable genotoxic and cytotoxic effects on cerebral endothelial cells, which might contribute to the progression of tissue damage in the central nervous system.

Topics: Animals; Apoptosis; Blotting, Western; Cells, Cultured; Chromosome Aberrations; Endothelium, Vascular; Glucose; Hypoxia; L-Lactate Dehydrogenase; Micronuclei, Chromosome-Defective; Naphthoquinones; Oxidative Stress; Rats; Reperfusion Injury; Swine; Telencephalon; Time Factors; Tumor Suppressor Protein p53

Rat liver epithelial cells were exposed to three quinones with different properties: menadione (2-methyl-1,4-naphthoquinone, vitamin K3), an alkylating as well as redox-cycling quinone, the strongly alkylating p-benzoquinone (BQ), and the non-arylating redox-cycler, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). All three quinones induced the activation of extracellular signal-regulated kinase (ERK) 1 and ERK 2 via the activation of epidermal growth factor receptor (EGFR) and MAPK/ERK kinases (MEK) 1/2. ERK activation resulted in phosphorylation at Ser-279 and Ser-282 of the gap junctional protein, connexin-43, known to result in the loss of gap junctional intercellular communication. Another EGFR-dependent pathway was stimulated, leading to the activation of the antiapoptotic kinase Akt via phosphoinositide 3-kinase. The activation of EGFR-dependent signaling by these quinones was by different mechanisms: (i) menadione, but not BQ or DMNQ, inhibited a protein-tyrosine phosphatase regulating the EGFR, as concluded from an EGFR dephosphorylation assay; (ii) although menadione-induced activation of ERK was unimpaired by pretreatment of cells with N-acetyl cysteine, activation by BQ and DMNQ was prevented; (iii) cellular glutathione (GSH) levels were strongly depleted by BQ. The mere depletion of GSH by application of diethyl maleate EGFR-dependently activated ERK and Akt, thus mimicking BQ effects. GSH levels were only moderately decreased by menadione and not affected by DMNQ. In summary, EGFR-dependent signaling was mediated by protein-tyrosine phosphatase inactivation (menadione), GSH depletion (BQ), and redox-cycling (DMNQ), funneling into the same signaling pathway.

Topics: Animals; Benzoquinones; Blotting, Western; Cell Line; Connexin 43; Dose-Response Relationship, Drug; Epithelial Cells; ErbB Receptors; Gap Junctions; Glutathione; HeLa Cells; Humans; Immunohistochemistry; Indicators and Reagents; Liver; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Models, Biological; Naphthoquinones; Oxidation-Reduction; Phosphorylation; Precipitin Tests; Rats; Signal Transduction; Time Factors; Ultraviolet Rays; Vitamin K 3

The presence of hypoxic cells in solid tumors has long been considered a problem in cancer treatment. Resistance of hypoxic cells to ionizing radiation and anticancer drugs has in part been attributed to changes in altered gene expression by hypoxia. We previously reported an activation of heat shock factor (Hsf) in murine tumor RIF cells following hypoxia and suggested that a subsequent accumulation of heat shock protein(s) (Hsp) is likely to contribute to the malignant progression of hypoxic tumor cells (Baek et al., 2001). In this study, we showed that hypoxia induced a DNA-binding activity of Hsf and activation of hsp70 gene expression in colon cancer Clone A cells, and that a naphthazarin derivative, S64, significantly inhibited the hypoxia-inducible hsp70 gene expression in Clone A cells. We also showed that S64 significantly reduced the cellular glutathione levels in this cell line. Considering the proposed effects of Hsp and glutathione on radiation and chemotherapy sensitivity, we suggest that the inhibitory effects of S64 on Hsf activation and cellular glutathione levels have potentially important clinical implications. We believe that the previously reported in vitro and in vivo anti-tumor effect of S64 (Song et al., 2000a, 2001) might be attributed, at least in part, to its effect on Hsf activation and/or glutathione depletion. We also believe that the detailed molecular mechanisms underlying the effects of S64 on Hsf and glutathione level following hypoxia deserve a more rigorous future study, the results of which could offer novel strategy to manipulate the resistance mechanisms of solid tumors.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Cell Line, Tumor; Cell Nucleus; DNA; Glutamate-Cysteine Ligase; Glutathione; HSP70 Heat-Shock Proteins; Hypoxia; Mice; Naphthoquinones; Protein Binding; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; RNA; Time Factors

In A549 cell culture, significant variability was found in sensitivity to actinomycin D. Using limiting dilution, actinomycin D-susceptible (G4S) and -resistant (D3R) subclones were isolated. G4S cells were also susceptible to protein synthesis inhibitors, a redox cycling quinone, and an electrophile with concomitant activation of caspases 3 and 9. D3R cells were resistant to these agents without caspase activation. Antioxidant profiles revealed that D3R cells had significantly higher glutathione and glutathione reductase activity but markedly lower catalase, glutathione peroxidase, and aldehyde reductase activities than G4S cells. Thus A549 cells contain at least two distinct subpopulations with respect to predisposition to cell death and antioxidant profile. Because sensitivities to agents and the antioxidant profile were inconsistent, mechanisms independent of antioxidants, including the apparent inability to activate caspases in D3R cells, may play an important role. Regardless, the results suggest that antioxidant profiles of asymmetrical cell populations cannot predict sensitivity to oxidants and warn that the use of single subclones is advisable for mechanistic studies using A549 or other unstable cell lines.

Topics: Adenocarcinoma; Aldehydes; Anisomycin; Antioxidants; Apoptosis; Caspases; Cytotoxins; Dactinomycin; Genetic Heterogeneity; Glutathione; Growth Inhibitors; Humans; Hydrogen Peroxide; Lung Neoplasms; Male; Naphthoquinones; Oxidation-Reduction; Oxidative Stress; Protein Synthesis Inhibitors; Superoxide Dismutase; Tumor Cells, Cultured

Reactive oxygen species (ROS) have been shown to be involved in the reduction of glomerular filtration rate observed after gentamicin (Genta) treatment in vivo, a phenomenon directly related with mesangial cell (MC) contraction. Our previous study reported that Genta induces concentration-dependent MC contraction and proliferation in vitro.. To study the possible mediation of ROS in the effect of Genta, ROS production was measured in primary cultures of rat MC stimulated with Genta (10-5 mol/L). In addition, the MC response to Genta in the presence of the ROS scavengers superoxide dismutase (SOD) and catalase (CAT) was studied. MC activation and O2- production were studied in the presence of an inhibitor of the NADP(H) oxidase, diphenylene iodinium (DPI), and in the presence of L-NAME, an inhibitor of nitric oxide synthases (NOS). Finally, the effects of Genta on SOD activity and mRNA expression were examined.. Genta (10-5 mol/L) induced an increase in O2- production and SOD activity that was neither accompanied by an elevation in cytosolic Cu/Zn-SOD mRNA expression nor by H2O2 accumulation. Genta induced MC contraction and proliferation that were inhibited by SOD plus CAT. Both the extracellular and intracellular ROS donor systems, xantine+xantine oxidase (X+XO) and dimethoxinaphtoquinone (DMNQ), respectively, also stimulated MC contraction and proliferation. Genta-induced MC activation and O2- production were inhibited by DPI. Genta-induced O2- production was inhibited by L-NAME. Furthermore, Genta did not induce detectable changes in membrane fluidity and lipid peroxidation.. These results strongly suggest that an oxidative-mediated pathway exists in Genta-induced MC activation. A portion of the production of O2- may be due to NADP(H) oxidase and NOS activation. The amount of ROS produced, rather than having a toxic effect, might play a role as a mediator of Genta-induced MC activation

Topics: Animals; Anti-Bacterial Agents; Catalase; Cell Division; Cell Membrane; Cells, Cultured; Female; Gene Expression Regulation, Enzymologic; Gentamicins; Glomerular Mesangium; Hydrogen Peroxide; Lipid Peroxidation; Membrane Fluidity; NADPH Oxidases; Naphthoquinones; Nitric Oxide Synthase; Rats; Rats, Wistar; Reactive Oxygen Species; RNA, Messenger; Superoxide Dismutase

We examined whether superoxide (O(2)(-)) is produced as a precursor of hydrogen peroxide (H(2)O(2)) in cultured thyroid cells using the cytochrome c method and the electron paramagnetic resonance (EPR) method. No O(2)(-) or its related radicals was detected in thyroid cells under the physiological condition. The presence of quinone, 2,3-dimethoxy-l-naphthoquinone (DMNQ), or 2-methyl-1, 4-naphthoquinone (menadione), in the medium produced O(2)(-) and hydroxyl radicals (OH*); the amount of H(2)O(2) generation was also increased. Incubation of follicles with DMNQ or menadione inhibited iodine organification (a step of thyroid hormone formation) and its catalytic enzyme, thyroid peroxidase (TPO). This inhibition should be caused by reactive oxygen species because the two quinones, particularly DMNQ, exert their effect through the generation of reactive oxygen species. It is speculated that the site-specific inactivation of TPO might have occurred at the heme-linked histidine residue of the TPO molecule, a critical amino acid for enzyme activity because OH* (vicious free radicals) can be formed at the iron-linked amino acid. TPO mRNA level and electrophoretic mobility of TPO were not inhibited by quinones. Our study suggests that thyroid H(2)O(2) is produced by divalent reduction of oxygen without O(2)(-) generation. If thyroid cells happen to be exposed to significant amount of reactive oxygen species, TPO and subsequent thyroid hormone formation are inhibited.

Topics: Animals; Cells, Cultured; Hydrogen Peroxide; Iodide Peroxidase; Naphthoquinones; Reactive Oxygen Species; Superoxides; Swine; Thyroid Gland; Vitamin K 3

The modulation of cell signaling by nitric oxide (NO) and superoxide (O(-)(2)) is associated with apoptotic cell death in inflammatory kidney diseases. Recently, we have shown that NO induces ceramide production in glomerular mesangial and endothelial cells and the ratio of NO and O(-)(2) determines whether cells live or die.. Glomerular endothelial and mesangial cells were labeled with [(14)C]serine, the precursor of all sphingolipids, then stimulated with reactive oxygen species- or reactive nitrogen species-generating substances and subjected to lipid extraction. Radioactive lipids were separated and analyzed by thin-layer chromatography. DNA fragmentation, as a characteristic feature of apoptosis, was measured by a nucleosome/DNA-ELISA, which quantitatively recorded the histone-associated DNA fragments.. Exposure of glomerular endothelial and mesangial cells to either NO donors or superoxide-generating substances led to a delayed and sustained ceramide formation that paralleled the induction of apoptosis in both cell types. Coincubation of endothelial cells with NO and superoxide, which led to the generation of peroxynitrite, caused a synergistic enhancement of ceramide generation and apoptosis when compared to either stimulus alone. By contrast, in glomerular mesangial cells costimulation with superoxide neutralized not only NO-induced apoptosis but also NO-induced ceramide formation, although O(-)(2) alone triggered ceramide formation in mesangial cells and caused cell death. Moreover, SIN-1, a substance that simultaneously releases NO and O(-)(2) and thereby generates peroxynitrite, also stimulated a delayed ceramide formation in endothelial cells but not in mesangial cells. Furthermore, exposure of endothelial cells to glucose oxidase, which generates hydrogen peroxide, or to exogenous hydrogen peroxide, also showed a dose-dependent increase in ceramide formation and apoptosis, although to a lesser extent than did superoxide.. These data suggest that ceramide represents an important mediator of reactive oxygen and nitrogen species-triggered cell responses, like apoptosis. There seem to be cell type-specific protective mechanisms that critically depend on a fine-tuned redox balance between reactive nitrogen and oxygen species to determine whether a cell undergoes apoptosis or survives when exposed to oxidative and/or nitrosative stress conditions.

Topics: Animals; Apoptosis; Cattle; Cells, Cultured; Ceramides; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Synergism; Kidney Glomerulus; Molsidomine; Naphthoquinones; Nitric Oxide; Nitric Oxide Donors; Spermine; Superoxides

Though reactive oxygen species (ROS) has been noticed to be involved in arsenic trioxide (As(2)O(3))-induced apoptosis of tumor cells, its role in apoptosis signaling remained to be elucidated. The objective of this work was to explore the association of the inherent cellular ROS level with the susceptibility of the tumor cells to apoptosis induction by As(2)O(3). Low concentration of As(2)O(3) was administered to cultured leukemic cell lines NB4, U937, HL60 and K562. The difference in apoptotic sensitivity was displayed among four cell types. ROS probes were incubated with the cells in the absence of As(2)O(3), and ROS was thus quantified relatively by flow cytometry. We manifested, in four cell types, the inherently existed difference in whole ROS quantity, and a positive correlation between the inherent ROS level and their apoptotic sensitivity to As(2)O(3). Furthermore, by interference using a ROS producer, we demonstrated that an elevation of ROS level would sensitize the cells to As(2)O(3)-induced apoptosis. The results of the present work suggested that the inherent ROS level might be determinative in tumor cells for their apoptotic susceptibility to As(2)O(3).

Topics: Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Cell Separation; Dose-Response Relationship, Drug; Flow Cytometry; Humans; In Situ Nick-End Labeling; Leukemia, Myeloid; Naphthoquinones; Oxides; Reactive Oxygen Species; Tumor Cells, Cultured

To investigate whether ascorbic acid could enhance the efficacy of arsenic trioxide (As(2)O(3)) combined with 2, 3-dimethoxy-1, 4-naphthoquinone (DMNQ) in inducing the apoptosis of leukemia cell line U937 and its possible mechanism.. Flow cytometry and electron microscopy were applied to detect apoptosis of U937 cells after treatment with various combinations of As(2)O(3), DMNQ and ascorbic acid for 24 hours.. As(2)O(3) and DMNQ induced-apoptosis of U937 cells was enhanced (35.24%-->61.20%) upon cotreatment with ascorbic acid. Catalase could reverse this effect of DMNQ. Ascorbic acid had no effect on DMNQ-induced apoptosis of U937 cells.. Ascorbic acid enhanced the apoptosis of U937 cells via reactive oxygen species-dependent pathway in the presence of As(2)O(3).

Topics: Apoptosis; Arsenic Trioxide; Arsenicals; Ascorbic Acid; Drug Synergism; Flow Cytometry; Humans; Naphthoquinones; Oxides; U937 Cells

Various analogues of 5,8-dimethoxy-1,4-naphthoquinone (DMNQ) such as 2- or 6-(1-hydroxyiminoalkyl)-DMNQs were prepared and evaluated for the antitumor action. (1-Hydroxyiminoalkyl)-DMNQ derivatives expressed greater antitumor action than (1-hydroxyalkyl)- or acyl-DMNQ derivatives. Moreover, 6-(1-hydroxyiminoalkyl)-DMNQ derivatives expressed higher antitumor action than 2-sudstituted ones, suggestive of a steric effect. Some of 6-(1-propyloxyalkyl)-DMNQ derivatives with an alkyl group of butyl to octyl moiety showed T/C values of >400%

Topics: Animals; Antineoplastic Agents; Drug Screening Assays, Antitumor; Male; Mice; Mice, Inbred ICR; Naphthoquinones; Sarcoma 180; Tumor Cells, Cultured

Topics: Animals; Aorta; Cattle; Cells, Cultured; Cyclic N-Oxides; Cyclosporine; Endothelium, Vascular; Enzyme Inhibitors; Flow Cytometry; Fluorescent Dyes; Free Radical Scavengers; Humans; Immunosuppressive Agents; Models, Biological; Naphthoquinones; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Oxidants; Spin Trapping; Superoxide Dismutase; Superoxides

Recent evidence suggests that the sphingolipid-derived second messenger ceramide and oxidative stress are intimately involved in apoptosis induction. Here we report that exposure of microcapillary glomerular endothelial cells to superoxide-generating substances, including hypoxanthine/xanthine oxidase and the redox cyclers DMNQ and menadione results in a dose-dependent and delayed increase in the lipid signaling molecule ceramide. Long-term incubation of endothelial cells for 2-30 h with either DMNQ or hypoxanthine/xanthine oxidase leads to a continuous increase in ceramide levels. In contrast, short-term stimulation for 1 min up to 1 h had no effect on ceramide formation. The DMNQ-induced delayed ceramide formation is dose-dependently inhibited by reduced glutathione, whereas oxidized glutathione was without effect. Furthermore, N-acetylcysteine completely blocks DMNQ-induced ceramide formation. All superoxide-generating substances were found to dose-dependently trigger endothelial cell apoptosis. In addition, glutathione and N-acetylcysteine also prevented superoxide-induced apoptosis and implied that ceramide represents an important mediator of superoxide-triggered cell responses like apoptosis.

Topics: Acetylcysteine; Animals; Antioxidants; Apoptosis; Cattle; Cells, Cultured; Ceramides; Dose-Response Relationship, Drug; Endothelium, Vascular; Glutathione; Hypoxanthine; Kidney Glomerulus; Naphthoquinones; Superoxides; Vitamin K; Xanthine Oxidase

Various anti-platelet drugs, including quinones, are being investigated as potential treatments for cardiovascular disease because of their ability to prevent excessive platelet aggregation. In the present investigation 3 naphthoquinones (2,3-dimethoxy-1,4-naphthoquinone [DMNQ], menadione, and 1,4-naphthoquinone [4-NQ]) were compared for their abilities to inhibit platelet aggregation, deplete glutathione (GSH) and protein thiols, and cause cytotoxicity. Platelet-rich plasma, isolated from Sprague-Dawley rats, was used for all experiments. The relative potency of the 3 quinones to inhibit platelet aggregation, deplete intracellular GSH and protein thiols, and cause cytotoxicity was 1,4-NQ > menadione >> DMNQ. Experiments using 2 thiol-modifying agents, dithiothreitol (DTT) and 1-chloro-2,4-dintrobenzene (CDNB), confirmed the key roles for GSH in quinone-induced platelet anti-aggregation and for protein thiols in quinone-induced cytotoxicity. Furthermore, the anti-aggregative effects of a group of 12 additional quinone derivatives were positively correlated with their ability to cause platelet cytotoxicity. Quinones that had a weak anti-aggregative effect did not induce cytotoxicity (measured as LDH leakage), whereas quinones that had a potent anti-aggregative effect resulted in significant LDH leakage (84-96%). In one instance, however, p-chloranil demonstrated a potent anti-aggregative effect, but did not induce significant LDH leakage. This can be explained by the inability of p-chloranil to deplete protein thiols, even though intracellular GSH levels decreased rapidly. These results suggest that quinones that deplete GSH in platelets demonstrate a marked anti-aggregative effect. If this anti-aggregative effect is subsequently followed by depletion of protein thiols, cytotoxicity results.

Topics: Animals; Blood Platelets; Cell Survival; Chloranil; Dinitrochlorobenzene; Dithiothreitol; Female; Glutathione; L-Lactate Dehydrogenase; Naphthoquinones; Platelet Aggregation; Proteins; Rats; Rats, Sprague-Dawley; Sulfhydryl Compounds; Vitamin K

The human colon carcinoma cell line Caco-2 was exposed to the oxidative stress-inducing agents menadione (MEN), 2,3-dimethoxy-1,4-naphthoquinone, and hydrogen peroxide. All three agents caused DNA damage which was assessed by alkaline unwinding. Further, all three agents induced intensive NAD+ depletion, followed by a decrease in intracellular ATP and viability. Inhibition of poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30) by 3-aminobenzamide prevented the depletion of NAD+. These cells had a higher viability and ATP content. The most pronounced effect was observed with 25 microM of MEN, while at higher levels a partial preservation of NAD+ was observed with no effect on ATP or viability. The chelation of intracellular calcium by bis-(o-aminophenoxy)-ethane-N,N,N1,N1-tetraacidic acid/tetraacetoxymethyl) ester also prevented the dramatic loss of NAD+, demonstrating that Ca2+ is an activating factor in PARP-mediated cell killing.

Topics: Adenosine Triphosphate; Benzamides; Caco-2 Cells; Calcium; Cell Death; Chelating Agents; DNA Damage; DNA, Neoplasm; Egtazic Acid; Glutathione; Humans; Hydrogen Peroxide; Kinetics; NAD; Naphthoquinones; Oxidants; Oxidative Stress; Poly(ADP-ribose) Polymerase Inhibitors; Vitamin K

The human colon carcinoma cell lines Caco-2 and HT-29 were exposed to three structurally related naphthoquinones. Menadione (MEN), 1,4-naphthoquinone (NQ), and 2,3-dimethoxy-1,4-naphthoquinone (DIM) redoxcycle at similar rates, NQ is a stronger arylator than MEN, and DIM does not arylate thiols. The Caco-2 cell line was particularly vulnerable to NQ and MEN and displayed moderate toxic effects of DIM. The HT-29 cell line was only vulnerable to NQ and MEN after inhibition of DT-diaphorase (DTD) with dicoumarol, whereas dicoumarol did not affect the toxicity of quinones to Caco-2 cells. DTD activity in the HT-29 and Caco-2 cell lines, as estimated by the dicoumarol-sensitive reduction of 2,6-dichlorophenolindophenol, was 393.7 +/- 46.9 and 6.4 +/- 2.2 nmol NADPH x min(-1) x mg protein(-1), respectively. MEN depleted glutathione to a small extent in the HT-29 cell line, but a rapid depletion similar to Caco-2 cells was achieved when dicoumarol was added. The data demonstrated that the DTD-deficient Caco-2 cell line was more vulnerable to arylating or redoxcycling quinones than DTD-expressing cell lines. Exposure of the Caco-2 cell line to quinones produced a rapid rise in protein disulphides and oxidised glutathione. In contrast to NQ and DIM, no intracellular GSSG was observed with MEN. The relatively higher levels of ATP in MEN-exposed cells may account for the efficient extrusion of intracellular GSSG. The reductive potential of the cell as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction was only increased by MEN and not with NQ and DIM. We conclude that arylation is a major contributing factor in the toxicity of quinones. For this reason, NQ was the most toxic quinone, followed by MEN, and the pure redoxcycler DIM elicited modest toxicity in Caco-2 cells.

Topics: Adenosine Triphosphate; Cell Survival; Colonic Neoplasms; Dicumarol; Glutathione; Glutathione Disulfide; Humans; Kinetics; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; Neoplasm Proteins; Sulfhydryl Compounds; Tumor Cells, Cultured; Vitamin K

The dithiocarbamates are well known for their antioxidant properties and effects on cellular transcriptional events. For example, pyrrolidine dithiocarbamate (PDTC) is widely used as an inhibitor of nuclear factor kappa B (NFkappaB) and this, or related compounds may have therapeutic potential in inhibiting atherosclerosis. However, the precise molecular mechanisms through which PDTC could elicit antioxidant or cell signaling effects in a cellular setting remain unclear. Furthermore, the mechanisms for the effects of PDTC on NFkappaB are likely to involve inhibition of binding of the transcription factor to DNA rather than an effect on the activation process as first proposed. In relation to pharmacological applications of such compounds, little is known of their interaction with endothelial cells, the anticipated site of action for inhibition of vascular related diseases. Until recently, PDTC was generally classified as an antioxidant but evidence for pro-oxidant effects have been reported. In this study, we have addressed this issue in bovine aortic endothelial cells and identified two mechanisms through which PDTC can exert antioxidant effects. At low concentrations (0-25 microM), PDTC induces a concentration dependent increase in cellular GSH levels through the increased activity of gamma-glutamylcysteine synthetase. At higher concentrations, GSH oxidation and apoptotic cell death occur. Using 2,3 dimethoxy-1,4-napthoquinone (DMNQ) as an intracellular generator of superoxide radicals, we find PDTC (10 microM) protects against the cytotoxicity of this agent through a GSH-independent mechanism.

Topics: Animals; Antioxidants; Apoptosis; Cattle; DNA; Endothelium, Vascular; Glutathione; Glutathione Disulfide; In Vitro Techniques; Naphthoquinones; NF-kappa B; Oxidation-Reduction; Oxidative Stress; Pyrrolidines; Superoxides; Thiocarbamates

Macrophages are a major source of cytokines and proinflammatory radicals such as superoxide. These mediators can be both produced and utilized by macrophages in autocrine-regulatory pathways. Therefore, we studied the potential role of oxygen radical-regulatory mechanisms in reprogramming macrophage apoptosis. Preactivation of RAW 264.7 cells with a nontoxic dose of the redox cycler 2,3-dimethoxy-1,4-naphthoquinone (5 microM) for 15 h attenuated S-nitrosoglutathione (1 mM)-initiated apoptotic cell death and averted accumulation of the tumor suppressor p53, which is indicative for macrophage apoptosis. Preactivation with superoxide promoted cyclooxygenase-2 induction that was NF-kappa B and AP-1 mediated. NF-kappa B activation was confirmed by p50/p65-heterodimer formation, I kappa B-alpha degradation, and stimulation of a NF-kappa B luciferase reporter construct. Furthermore, a NF-kappa B decoy approach abrogated cyclooxygenase-2 (Cox-2) expression as well as inducible protection. The importance of AP-1 for superoxide-mediated Cox-2 expression and cell protection was substantiated by using the extracellular signal-regulated kinase-inhibitor PD98059 and the p38-inhibitor SB203580, which blocked Cox-2 expression. In corroboration, Cox-2 expression was hindered by a dominant-negative c-jun mutant (TAM67). Protection from apoptosis was verified in human macrophages with the notion that superoxide promoted Cox-2 expression, which in turn attenuated nitric oxide-evoked caspase activation. We conclude that the sublethal generation of oxygen radicals reprograms macrophages by NF-kappa B and AP-1 activation. The resulting hyporesponsiveness reveals an attenuated apoptotic program in association with Cox-2 expression.

Topics: Adjuvants, Immunologic; Animals; Apoptosis; Cell Line; Cyclooxygenase 2; Dose-Response Relationship, Drug; Isoenzymes; Macrophages; Mice; Naphthoquinones; NF-kappa B; Nitric Oxide; Prostaglandin-Endoperoxide Synthases; Superoxides; Transcription Factor AP-1; Tumor Suppressor Protein p53

gamma-Glutamyl transpeptidase (GGT) plays an important role in glutathione (GSH) metabolism. GGT expression is increased in oxidant-challenged cells; however, the signaling mechanisms involved are uncertain. The present study used 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), a redox cycling quinone that continuously produced H2O2 in rat lung epithelial L2 cells. It was found that DMNQ increased GGT mRNA content by increasing transcription, as measured by nuclear run-on. This was accompanied by increased GGT specific activity. Cycloheximide, a protein synthesis inhibitor, blocked neither the increased GGT mRNA content nor the increased GGT transcription rate caused by DMNQ, suggesting that increased GGT transcription was a direct rather than secondary response. Previous data from this laboratory (R.-M. Liu, H. Hu, T. W. Robinson, and H. J. Forman. Am. J. Respir. Cell Mol. Biol. 14: 186-191, 1996) showed that tert-butylhydroquinone (TBHQ) increased GGT mRNA content by increasing its stability. TBHQ differs markedly from DMNQ in terms of its conjugation with GSH and H2O2 generation. Together, the data suggest that quinones upregulate GGT through multiple mechanisms, increased transcription and posttranscriptional modulation, which are apparently mediated through generation of reactive oxygen species and GSH conjugated formation, respectively.

Topics: Animals; Antioxidants; Cells, Cultured; Epithelial Cells; gamma-Glutamyltransferase; Hydrogen Peroxide; Hydroquinones; Lung; Naphthoquinones; Oxidation-Reduction; Rats; Reactive Oxygen Species; Superoxides

The formation of nitric oxide (NO.) and superoxide (O2-) promotes rat mesangial cell death. Apoptotic death is characterized by DNA fragmentation, caspase-3 activation and concomitant poly(ADPribose) polymerase cleavage, as well as accumulation of the tumor suppressor protein p53. In close association with apoptotic parameters we noticed upregulation of heme oxygenase by the NO donor S-nitrosoglutathione (GSNO) and the redox cycler 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) in a time- and concentration-dependent manner. In response to the NO. donor, heme oxygenase-1 expression was more easily obtained than initiation of apoptosis. Radical (NO./O2-) cogeneration abrogated DNA fragmentation, suppressed caspase activation and lowered p53 accumulation, thereby promoting cell survival of mesangial cells. In contrast, heme oxygenase-1 expression remained elevated under conditions of GSNO/DMNQ coadministration. Conclusively, heme oxygenase-1 is a stress marker for both nitrosative and oxidative stress. Accumulation of heme oxygenase-1 is found under conditions of both, apoptotic cell death and cell survival, thereby questioning a specific cytoprotective role of heme oxygenase-1 under conditions of NO. and/or O2- formation in rat mesangial cells.

Topics: Animals; Apoptosis; Cells, Cultured; DNA Fragmentation; Enzyme Induction; Glomerular Mesangium; Glutathione; Heme Oxygenase (Decyclizing); Naphthoquinones; Neuroprotective Agents; Nitric Oxide; Nitroso Compounds; Oxidative Stress; Rats; S-Nitrosoglutathione; Tumor Suppressor Protein p53; Up-Regulation

Our previous studies demonstrated that menadione is cytotoxic to rat platelets. In an attempt to assess the relative contributions of enzymatic redox cycling versus arylation in menadione-induced cytotoxicity, we have studied three quinones with different mechanisms of action: 2,3-dimethoxy-1,4-naphthoquinone (DMNQ; pure redox cycler), menadione (both redox cycler and arylator), and 1,4-benzoquinone (BQ; pure arylator). BQ was more toxic to rat platelets than menadione, while DMNQ did not cause LDH leakage at all. Cellular uptake kinetics revealed that DMNQ concentration taken up by the cells was equivalent to that decreased in incubation medium. On the other hand, the concentrations of BQ and menadione taken into the cells were significantly lower than the decreases in concentrations seen in the incubation medium. This suggests indirectly that BQ and menadione may have undergone arylation, binding to glutathione (GSH) or protein thiols. The difference in arylation capacity between BQ and menadione was well correlated with their relative cytotoxicity (LDH leakage) observed in platelets. All three quinones caused a rapid, extensive depletion of intracellular GSH in platelets. Treatments with BQ and menadione did not result in formation of oxidized glutathione (GSSG), whereas DMNQ showed a time-dependent increase in GSSG. Altogether, these results suggest that enzymatic redox cycling does not play a critical role in quinone-induced cytotoxicity in rat platelets, while arylation is likely to be quinone's primary mechanism of action.

Topics: Alkylation; Animals; Benzoquinones; Blood Platelets; Cell Survival; Chromatography, High Pressure Liquid; Female; Glutathione; Homeostasis; L-Lactate Dehydrogenase; Naphthoquinones; Oxidative Stress; Oxygen Consumption; Quinones; Rats; Rats, Sprague-Dawley; Vitamin K

Access of intracellular Ags SSA/Ro and SSB/La to cognate maternal autoantibodies is unexplained despite their strong association with congenital heart block. To investigate the hypothesis that apoptosis facilitates surface accessibility of these Ags, human fetal cardiac myocytes from 16- to 22-wk abortuses were established in culture using a novel technique in which cells were isolated after perfusing the aorta with collagenase. Confirmation of cardiac myocytes included positive staining with antisarcomeric alpha-actinin and contractility induced by 1.8 mM calcium. Incubation with 0.5 microM staurosporine or 0.3 mM 2,3-dimethoxy-1,4-naphthoquinone induced the characteristic morphologic and biochemical changes of apoptosis. The cellular topology of Ro and La was evaluated with confocal microscopy and determined in nonapoptotic and apoptotic cardiocytes by indirect immunofluorescence. In permeabilized nonapoptotic cardiocytes, Ro and La were predominantly nuclear, and propidium iodide (PI) stained the nucleus. In early apoptotic cardiocytes, condensation of the PI- and Ro- or La-stained nucleus was observed, accompanied by Ro/La fluorescence around the cell periphery. In later stages of apoptosis, nuclear Ro and La staining became weaker, and PI demonstrated nuclear fragmentation. Ro/La-stained blebs emerged from the cell membrane, a finding observed in nonpermeabilized cells, supporting an Ab-Ag interaction at the cell surface. In summary, induction of apoptosis in cultured cardiocytes results in surface translocation of Ro/La and recognition by Abs. Although apoptotic cells are programmed to die and do not characteristically evoke inflammation, binding of maternal Abs and subsequent influx of leukocytes could damage surrounding healthy fetal cardiocytes.

Topics: Antigen-Antibody Reactions; Antigens, Surface; Aorta; Apoptosis; Autoantibodies; Autoantigens; Autoimmune Diseases; Biological Transport; Cell Nucleus; Cell Separation; Cells, Cultured; Collagenases; Enzyme Inhibitors; Female; Fetal Heart; Fetal Proteins; Heart Block; Humans; Immunity, Maternally-Acquired; Muscle Proteins; Myocardium; Naphthoquinones; Pregnancy; Pregnancy Complications; Protein Isoforms; Ribonucleoproteins; RNA, Small Cytoplasmic; SS-B Antigen; Staurosporine

gamma-Glutamylcysteine synthetase (GCS) is the initial and rate-limiting enzyme in the glutathione (GSH) de novo synthesis pathway. GCS is composed of a heavy (73-kDa) catalytic subunit and a light (30-kDa) regulatory subunit, which maintains the Km for glutamate near physiologic concentrations. Previous studies have shown that the steady-state mRNA level and gene transcription for the catalytic subunit increased in response to the redox-cycling quinone 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) in rat lung epithelial L2 cells (M. M. Shi, et al., 1994, J. Biol. Chem. 269,26512-26517). The ratio of the catalytic to regulatory subunit mRNAs varies among tissues, and the anticancer drug cisplatin appears to induce only the catalytic subunit, suggesting independent gene regulation of the two subunits. Nonetheless, the present study found that the steady-state mRNA level and the transcription rate of the GCS regulatory subunit also increased under DMNQ-induced oxidative stress. Changes in mRNA followed a pattern similar to that for the catalytic subunit. The mRNA levels of the two subunits of GCS also both increased above the baseline levels in cells treated with BSO, an inhibitor of GCS enzymatic activity. These data suggest that, under conditions of oxidative stress or glutathione depletion, the regulatory subunit is upregulated at the level of mRNA transcription. Along with the elevation of the catalytic subunit, this increase in GCS regulatory subunit transcription contributes to increases in GCS enzymatic activity and cellular GSH content.

Topics: Animals; Buthionine Sulfoximine; Cell Line; Epithelium; Glutamate-Cysteine Ligase; Glutathione; Lung; Naphthoquinones; Oxidative Stress; Rats; RNA, Messenger; Transcription, Genetic; Up-Regulation

During proliferative glomerulonephritis, the early phase of mesangiolysis is linked to increased nitric oxide (NO) production. NO. as well as superoxide (O2-) are inflammatory mediators that are generated by mesangial cells (MC) after cytokine stimulation. Added individually, both radicals induce MC apoptosis. However, the co-existence of a defined NO./O2- ratio is cross-protective. Apoptosis is characterized by specific features such as chromatin condensation, DNA strand breaks, and the occurrence of apoptotic regulating proteins. The tumor suppressor p53 and Bax (Bcl-2 associated protein x) are considered to be classical death promotors, which accumulate after toxic insults. To study p53 and Bax protein accumulation in NO. and/or O2(-)-induced apoptosis, we used the NO-donor S-nitrosoglutathione (GSNO) and the redox cycler 2,3-dimethoxy-1,4-naphtoquione (DMNQ). Both agonists initiated DNA fragmentation in a concentration dependent manner associated with transient p53 and Bax up-regulation. Co-generation of NO./O2- resulted not only in reduced DNA fragmentation, but also in decreased Bax accumulation. Comparable to the NO./O2- co-generation, cytokines failed to induce apoptosis. In contrast, cytokines in combination with pyrrolidine dithiocarbamate, which blocks endogenous superoxide dismutase, allowed p53 and Bax accumulation as well as DNA fragmentation. Our results demonstrate p53 and Bax as early components in NO. and O2(-)-induced rat MC apoptosis and point to the NO./O2- interaction as a naturally occurring cell defense mechanism.

Topics: Animals; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Biotin; Cells, Cultured; Chromosomes; Cytokines; Deoxyuracil Nucleotides; DNA Fragmentation; Glomerular Mesangium; Glutathione; Naphthoquinones; Nitric Oxide; Nitroso Compounds; Oxygen; Platelet Aggregation Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Pyrrolidines; Rats; S-Nitrosoglutathione; Staining and Labeling; Superoxide Dismutase; Superoxides; Thiocarbamates; Time Factors; Tumor Suppressor Protein p53; Up-Regulation

Menadione (2-methyl-1,4-naphthoquinone, a redox cycling and arylating quinone; 5-100 microM) inhibited the canalicular vacuolar accumulation (CVA) of a fluorescent bile acid, cholyl-lysyl-fluorescein (CLF), in rat hepatocyte couplets. This was associated with depletion of reduced glutathione and accumulation of oxidized glutathione, the latter indicating that the concentrations of menadione used were able to induce oxidative stress. There was no associated cytotoxicity as indicated by ATP content. Treatment of couplets with the redox cycling quinone 2,3-dimethoxy-1,4-naphthoquinone (up to 100 microM) had relatively little effect on CVA, suggesting that the magnitude of reactive oxygen formation induced by this compound was insufficient to disrupt canalicular integrity. In comparison, the arylation of protein thiol groups by p-benzoquinone (up to 100 microM) proved to be more potent in inhibiting canalicular vacuolar accumulation. The predominant mechanism of menadione-induced inhibition of couplet hepatobiliary function is therefore more likely to involve the arylation of critical thiol groups (such as those in the F-actin cytoskeleton) rather than their oxidation. The oxidative effects of menadione could, however, potentiate the deleterious effects induced by arylation, such as by reduced glutathione depletion.

Topics: Adenosine Triphosphate; Animals; Benzoquinones; Biliary Tract; Cell Membrane; Cell Separation; Glutathione; Homeostasis; Liver; Naphthoquinones; Oxidation-Reduction; Rats; Vacuoles; Vitamin K

The role of mitochondrial dysfunction in the mechanisms of cell killing by quinones of differing chemical reactivities was investigated. Freshly isolated hepatocyte suspensions were exposed to 2,3-dimethoxy-1,4-naphthoquinone, 2-methyl-1,4-naphthoquinone, 1,4-naphthoquinone or 1,4-benzoquinone in the presence or absence of cyclosporine A, ruthenium red, fructose or the combination of fructose plus oligomycin. All of the quinones caused concentration-dependent cell killing as assessed by the leakage of lactate dehydrogenase. However, only 2,3-dimethoxy- and 2-methyl-naphthoquinone caused a depolarization of mitochondrial membrane potential; cell killing by 1,4-naphthoquinone or 1,4-benzoquinone was not accompanied by mitochondrial depolarization. Neither cyclosporine A nor ruthenium red protected against cell killing or loss of mitochondrial membrane potential caused by any of the quinones examined. In contrast, fructose protected cells against all four quinones. For the redox cycling naphthoquinones, oligomycin reversed the protection afforded by fructose. However, the cytoprotective effect of fructose against the arylating quinones, 1,4-naphthoquinone and 1,4-benzoquinone, was not reversed by oligomycin. The results suggest that cell killing by redox cycling naphthoquinones is a manifestation of mitochondrial depolarization, not ATP depletion. In contrast, the fructose-mediated protection from arylating quinones is consistent with ATP depletion being a critical event leading to cell death. According, although a vast array of quinone compounds are known to be cytotoxic, the mechanism of cell killing by individual members of this chemical class differs and is determined primarily by the chemical reactivity of the individual quinone.

Topics: Animals; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Liver; Male; Membrane Potentials; Mitochondria, Liver; Naphthoquinones; Rats; Rats, Sprague-Dawley; Vitamin K 3

Topics: Cell Line; DNA Damage; DNA, Neoplasm; Humans; Leukemia, Erythroblastic, Acute; NAD; Naphthoquinones; Oxidation-Reduction; Oxidative Stress; Tumor Cells, Cultured; Vitamin K

The in vitro testing of antitumor drugs involves the use of mouse and human tumor cells. In particular, there is interest in developing agents active against human solid tumors. We examined several biochemical parameters that may contribute to the differential sensitivity of the cell lines used in our laboratory to the toxic effects of antitumor compounds. The tumor cell lines examined were of mouse (colon 38, L1210 leukemia, and C1498 leukemia) and human origin (CEM leukemia, CX1 colon, H116 colon, HCT8 colon and H125 lung). Quinone reductase activity was markedly different between leukemia and solid-tumor cell lines of either mouse or human origin, with increased activity being observed in the solid-tumor cell lines relative to the leukemia lines. GSH transferase activity also was generally increased in solid-tumor relative to leukemia cell lines. Superoxide dismutase activity and thiol levels were similar in leukemia and solid-tumor cell lines, except that thiol levels were very low in colon 38. Mouse cell lines from in vitro passage had somewhat higher activity of superoxide dismutase and thiol levels than did cells maintained in vivo, indicating relatively increased antioxidant defenses. The toxicity of 2,3-dimethoxy-1,4-naphthoquinone, a model quinone that exerts its toxic effects via production of reactive oxygen species, was significantly lower in mouse lines maintained in vitro than in those tested in vivo, whereas the toxicity of another quinone, menadione, was just slightly lower. Quinone reductase activity, GSH transferase activity, and thiol levels were significantly higher in the human lines than in the mouse lines. Accordingly, the toxicity of both quinones tended to be lower in the human lines than in the mouse lines.

Topics: Animals; Antineoplastic Agents; Drug Screening Assays, Antitumor; Glutathione Transferase; Humans; Mice; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; Quinones; Superoxide Dismutase; Tumor Cells, Cultured; Vitamin K

A characteristic feature of many types of chemically induced oxidative stress is a depletion of the pyridine nucleotide NAD+. This has been attributed to either its hydrolysis to nicotinamide and ADP-ribose or to its phosphorylation (interconversion) to NADP+. In this study the exposure of rat hepatocytes to either tert-butyl hydroperoxide (250-750 microM) or 2,3-dimethoxy-1,4-naphthoquinone (50 microM) resulted in a rapid depletion of NAD+ with no change in the level of NAD. The depletion of NAD+ was accompanied by an increase in nicotinamide. The rate of NAD+ deletion induced by tert-butyl hydroperoxide (500 microM) and 2,3-dimethoxy-1,4-naphthoquinone (50 microM) was reduced by preincubating the hepatocytes for 1 hr with either 3-aminobenzamide (20 mM), nicotinamide (10 mM) or theophylline (7.5 mM), potent inhibitors of poly(ADP-ribose)polymerase. In cells exposed to 2,3-dimethoxy-1,4-naphthoquinone (50 microM) extensive oxidation of NADPH to NADP+ was observed; this was followed by an increase in the level of NADP(+) + NADPH (NADP(H)). However, no change in the total pyridine nucleotide (NAD(H) + NADP(H)) pool was detected. Exposure to tert-butyl hydroperoxide resulted in the oxidation of NADPH to NADP+ and a decrease in total pyridine nucleotide pool. These results suggest that during oxidative stress, NAD+ is hydrolysed to nicotinamide, possibly by the activation of poly(ADP-ribose)polymerase and that the depletion of NAD+ is independent of the increase in NADP+. Furthermore, no evidence of an interconversion of NAD+ to NADP+ was found.

Topics: Animals; Cells, Cultured; Enzyme Activation; Liver; Male; NAD; Naphthoquinones; Niacinamide; Oxidative Stress; Peroxides; Poly(ADP-ribose) Polymerase Inhibitors; Rats; Rats, Wistar; tert-Butylhydroperoxide

Quinone-induced cell death is often attributed to oxidative stress during which the formation of DNA strand breaks is thought to play an important role. In this study, extensive DNA damage was observed in human chronic myelogenous leukemic cells (K562) exposed for 15 minutes to low concentrations (15-100 microM) of the redox cycling quinones 2,3-dimethoxy-1,4-naphthoquinone (2,3-diOMe-1,4-NQ) and menadione. However, DNA strand breakage and cell death could not be attributed to oxidative stress as the intracellular level and redox status of the reducing equivalents NADP(H) and GSH were unaffected. The intracellular level of NAD+ was found to correlate well with the extent of DNA repair (r = 0.93, P < 0.02) and cell proliferation (r = 0.96, P < 0.01) in cells exposed to the quinones. In contrast, a significant decrease in the level of intracellular ATP was only observed in cells exposed to menadione (50-100 microM). These results suggest that redox cycling quinones are capable of inducing DNA damage in mammalian cells by a mechanism that does not involve oxidative stress. Following DNA damage, cell death is dependent on the availability of NAD+, which may be key to the rapid repair of strand breaks.

Topics: Adenosine Triphosphate; Cell Death; Cell Division; DNA Damage; DNA, Neoplasm; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; NAD; Naphthoquinones; Oxidation-Reduction; Oxidative Stress; Substrate Cycling; Tumor Cells, Cultured; Vitamin K

Light-induced FTIR QA-/QA difference spectra corresponding to the photoreduction of the primary quinone acceptor QA have been obtained for Rhodobacter sphaeroides RCs reconstituted with chainless symmetrical quinones in order to study the influence of the side chain and of molecular asymmetry on the binding of natural quinones to the QA site. The main vibrational modes of the quinones in vivo were obtained by analysis of the isotope effects induced by 18O substitution on the carbonyls and by comparison with the IR absorption spectra of the isolated quinones. For isolated 2,3-dimethoxy-5,6-dimethyl-1,4-benzoquinone (MQ0), 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone, DQ), and 2,3-dimethyl-1,4-naphthoquinone (DMNQ), the IR spectra together with mass spectroscopy data of partially 18O labeled quinones show that the labeling of one carbonyl leads to only a minor shift of the vibrational frequency of the opposite carbonyl. This observation demonstrates an essentially uncoupled behavior of the two C = O groups. Upon reconstitution of QA-depleted RCs with these symmetrical quinones, the double-difference spectra calculated from the QA-/QA spectra of the 18O-labeled and unlabeled quinones reveal a splitting of the quinone C = O modes. This splitting and the frequency downshift of the C = O vibrations upon binding to the QA site are comparable to those previously reported for the C = O modes of quinones containing an isoprenoid (Q8, Q6, Q1) or a phytyl chain (vitamin K1) [Breton, J., Burie, J.-R., Berthomieu, C., Berger, G., & Nabedryk, E. (1994) Biochemistry 33, 4953-4965]. This observation demonstrates that the replacement of the side chain by a methyl group does not impair the asymmetrical bonding interactions of the two quinone carbonyls with the protein. This asymmetry is traceable to the two distinct amino acid residues which have been proposed, on the basis of X-ray structural studies, to form hydrogen bonds with the carbonyls of the quinone. The close analogy between the double-difference spectra calculated for RCs reconstituted either with vitamin K1 or with DMNQ shows that the phytyl chain of vitamin K1 imparts no specific constraint on the geometry of the menaquinone head group in its binding site for both the neutral and the semiquinone state. In contrast, the double-difference spectra calculated for RCs reconstituted either with MQ0 or with Q6 (or Q1) exhibited significant differences in the relative amplitudes of the bands assigned to the mixed

Topics: Binding Sites; Hydrogen Bonding; Naphthoquinones; Photosynthetic Reaction Center Complex Proteins; Quinones; Rhodobacter sphaeroides; Spectrophotometry; Spectrophotometry, Infrared; Spectroscopy, Fourier Transform Infrared; Vitamin K 1

Glutathione (GSH) is one of the most important physiological antioxidants involved in detoxification of hydrogen peroxide and lipid hydroperoxide. Previous studies have shown that cells can maintain and even increase cellular GSH content in response to sublethal oxidative stress. We hypothesized that gamma-glutamylcysteine synthetase (gamma GCS), the rate-limiting enzyme in de novo GSH synthesis, could be induced by oxidative stress. Rat lung epithelial L2 cells were challenged with 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), generates O2.- and H2O2 continuously through redox cycling. Exposure of confluent L2 cells with sublethal doses of DMNQ caused sustained elevation of cellular GSH levels over a 24-h period (to 2.5-fold with 10 microM). DMNQ caused increases in gamma GCS activity (70% at 24 h with 10 microM), the gamma GCS catalytic heavy subunit (gamma GCS-HS) protein level, and gamma GCS-HS mRNA content (approximately 4-fold after 6 h with 10 microM). The elevation of gamma GCS-HS mRNA by DMNQ was eliminated by co-incubation with actinomycin D. Nuclear run-on experiments demonstrated that the transcriptional rate of the gamma GCS-HS gene was increased by 3- or 6-h exposure to 10 microM DMNQ. Our results suggested that the induction of de novo GSH synthesis by naphthoquinone-induced oxidative stress is associated with the transcriptional activation of the gamma GCS-HS gene and the subsequent elevation in gamma GCS activity. Unlike simpler quinones, DMNQ cannot form a GSH conjugate. Thus, the induction of gamma GCS-HS gene transcription does not require formation of an electrophile-glutathione conjugate.

Topics: Animals; Base Sequence; Cells, Cultured; Cycloheximide; Dactinomycin; Epithelium; Glutamate-Cysteine Ligase; Glutathione; Lung; Molecular Sequence Data; Naphthoquinones; Oxidative Stress; Rats; RNA, Messenger; Transcription, Genetic

Glutathione (GSH), an important physiological antioxidant, is synthesized de novo by the sequential reactions of gamma-glutamylcysteine synthetase (gamma GCS) and GSH synthetase. In the present studies, incubation with the quinones 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) and menadione (MQ), which generate superoxide and hydrogen peroxide, was used to investigate GSH synthesis in bovine pulmonary artery endothelial cells under oxidative stress. MQ can also cause initial depletion of GSH through conjugation, whereas DMNQ cannot. during continuous exposure to DMNQ (5 or 10 microM), elevation of GSH by DMNQ started after 6 h, almost doubled after 24 h, and remained at this level to 48 h. The elevation of GSH by DMNQ was mostly in the reduced form, and the ratio of reduced to oxidized glutathione remained unchanged for the first 24 h. Treatment with MQ (25 or 50 microM) for 30 min caused a significant decrease in GSH and total glutathione. After changing the medium to remove any residual MQ, GSH content doubled during the next 12 h. The enzymatic activity of gamma GCS, the rate-limiting enzyme of GSH biosynthesis, increased twofold after 12 h of exposure of cells to either 5 microM DMNQ or 50 microM MQ. Both DMNQ and MQ treatment caused concentration- and time-dependent increases in gamma GCS-mRNA expression. The elevation of gamma GCS-mRNA content by DMNQ for 12 h was completely blocked by coincubation with 0.05 microgram/ml actinomycin D but not 0.5 microgram/ml cycloheximide, suggesting the elevation of gamma GCS-mRNA content occurred through increased transcription. Our results suggest that increased de novo GSH synthesis, mediated by an elevation in gamma GCS, constitutes an adaptive response to oxidative stress.

Topics: Animals; Base Sequence; Benzoquinones; Cattle; Cells, Cultured; Cycloheximide; Dactinomycin; Endothelium, Vascular; Glutamate-Cysteine Ligase; Glutathione; Molecular Probes; Molecular Sequence Data; Naphthoquinones; Oxidative Stress; Pulmonary Artery; RNA, Messenger

Increasing concentrations (1-100 microM) of the redox cycling quinone, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), stimulated growth, triggered apoptosis, or caused necrosis of pancreatic RINm5F cells, depending on the dose and duration of the exposure. Following the exposure of RINm5F cells to 10 microM DMNQ, ornithine decarboxylase activity and polyamine biosynthesis increased. This was accompanied by enhanced cell proliferation. Conversely, exposure to 30 microM DMNQ for 3 h resulted in the inhibition of ornithine decarboxylase, intracellular polyamine depletion, and apoptotic cell killing. Pretreatment of the cultures with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, restored polyamine levels and prevented apoptosis. Exposure to the same DMNQ concentration for only 1 h, with subsequent re-incubation in growth medium, neither caused polyamine depletion nor resulted in apoptosis. Finally, exposure to an even higher DMNQ concentration (100 microM) for either 1 or 3 h caused rapid intracellular Ca2+ overload, ATP, NAD+, and glutathione depletion, and extensive DNA single strand breakage, which resulted in necrotic cell death. Our results show that a disturbance of polyamine biosynthesis occurred prior to cell growth or apoptosis elicited by oxidative stress. In addition, we show that effects as opposite as cell proliferation and deletion, by either apoptosis or necrosis, can be induced, in the same system, by varying the exposure to a prooxidant.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adenosylmethionine Decarboxylase; Animals; Apoptosis; Cell Division; Cell Line; Cell Survival; Deoxyguanosine; Dose-Response Relationship, Drug; Insulin; Insulin Secretion; Islets of Langerhans; Kinetics; Naphthoquinones; Necrosis; Oxidants; Polyamines; Tetradecanoylphorbol Acetate; Time Factors

Quinones are intracellular H2O2 generators that have been used extensively in models of oxidant injury; however, their toxicity is mediated partially through direct conjugation with glutathione (GSH). To focus upon the action of extracellular GSH in preventing H2O2-mediated toxicity, we used 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), which cannot conjugate with GSH but does continuously generate H2O2 through redox cycling. A eukaryotic cell line (3T3-GGT) stably overexpressing gamma-glutamyl transpeptidase (GGT) activity was used to study the role of GGT in utilizing extracellular GSH against DMNQ-induced oxidative stress. DMNQ (0 to 150 microM) caused a dose-dependent decrease of intracellular GSH and adenosine 5'-triphosphate (ATP) in both control and 3T3-GGT cells. The rate of H2O2 escape into the medium during DMNQ exposure was also the same in both cell lines. Administration of GSH helped to maintain intracellular GSH and supported resistance to ATP depletion caused by DMNQ in 3T3-GGT cells but not in control cells. The protective effect of extracellular GSH was completely prevented by acivicin, an inhibitor of GGT. Our results suggest that GGT-dependent breakdown of extracellular GSH for subsequent intracellular resynthesis helped to maintain cellular GSH levels and increased cellular resistance against DMNQ-induced oxidative injury.

Topics: 3T3 Cells; Animals; Blotting, Western; gamma-Glutamyltransferase; Gene Expression; Glutathione; Glycosylation; Humans; Hydrogen Peroxide; Isoxazoles; Mice; Naphthoquinones; Transfection

Our previous study demonstrated an increase in tyrosine protein phosphorylation and phosphatidylinositol phosphorylation induced by oxidants, suggesting a proliferative potential for these reactive substances. In the present study, we focus our investigation on the similarity between a redox cycling naphthoquinone and orthovanadate (VO), an oxidant generator as well as a potent phosphotyrosyl phosphatase inhibitor, in growth of 3T3-L1 cells cultured in serum-free media. Vanadate increased [3H]thymidine incorporation in a concentration-dependent manner. However, in the presence of insulin, epidermal growth factor, or platelet-derived growth factor, VO synergistically augmented, by as much as fivefold, DNA synthesis stimulated by these growth factors. An increase in the association of PI 3-kinase with the insulin receptor was also observed in cells treated with insulin + VO. Expression of the c-fos gene, a marker of early nuclear event in mitogenesis induced by insulin, EGF, and PDGF, but not vasopressin, a non-tyrosine kinase-linked mitogen, was also enhanced by VO. Flow cytometric analysis indicated an acceleration of cell cycle progression when VO and insulin were present simultaneously. Striking similarity was observed between VO and 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) in PI 3-kinase activation, c-fos proto-oncogene expression, and DNA synthesis, key events associated with cell growth. Additionally, both VO and DMNQ gave rise to DMPO-.OH adduct EPR signal measured with cell suspensions. These data support an oxidant-mediated increase in tyrosine protein phosphorylation early in the signal transduction cascade of growth factor receptors, leading to augmentation of cell proliferation.

Topics: 3T3 Cells; Animals; Cell Division; DNA; Electron Spin Resonance Spectroscopy; Epidermal Growth Factor; Gene Expression; Genes, fos; Growth Substances; Insulin; Mice; Naphthoquinones; Oxidation-Reduction; Phosphatidylinositol 3-Kinases; Phosphotransferases; Platelet-Derived Growth Factor; Receptor, Insulin; S Phase; Vanadates

In guinea pig and rat cardiac tissue, redox cycling benzoquinones (2,5-dimethyl-p-benzoquinone and duroquinone) and naphthoquinones (menadione and 2,3-dimethoxy-1,4-naphthoquinone) generated superoxide anion (O2-.) both through one- and two-electron reductions, the generation being significantly greater in guinea pig than in rat tissue. In electrically driven left atria isolated from guinea pig and rat, menadione and 2,5-dimethyl-p-benzoquinone but not duroquinone caused a concentration-dependent positive inotropic effect. Unlike guinea pig, 2,3-dimethoxy-1,4-naphthoquinone had no effect in rat tissue. Naphthoquinones and 2,5-dimethyl-p-benzoquinone were more active in guinea pig than in rat tissue, their effect being dependent on the release of catecholamines from adrenergic stores. A linear relationship (r = 0.90) between the amount of O2-. generated by benzo- and naphthoquinones in guinea pig and rat heart and the extent of catecholamine-dependent positive inotropic effect was evident. An amount of O2-. higher than 600 nmol/g of tissue per min was calculated to be necessary to determine the catecholamine-mediated increase in contractility. Lipid peroxidation was not involved in quinone-induced catecholamine release.

Topics: Animals; Atrial Function; Benzoquinones; Catecholamines; Cyclohexenes; Free Radicals; Guinea Pigs; Heart Atria; In Vitro Techniques; Lipid Peroxidation; Microsomes; Mitochondria, Heart; Myocardial Contraction; Naphthoquinones; Oxidation-Reduction; Rats; Superoxides; Vitamin K

Exposure of cells to thiol oxidizing agents can result in the modification of key proteins involved in cell signalling. Such changes have been shown to affect agonist-stimulated phosphoinositide metabolism, activation of protein kinases and intracellular Ca2+ signals, which result in abnormalities in cell metabolisms and growth. Here, we show that moderate levels of oxidants potentiate growth signals and either enhance cell proliferation or facilitate cell differentiation, whereas inhibition of growth signals by higher oxidant concentrations can block cell proliferation and activate programmed cell death (PCD). Finally, a general alteration of multiple signalling pathways associated with increased catabolic reactions results in cell death by necrosis. Our data suggest that oxidant interaction with cell signalling systems may exert opposite effects, depending on the dose, and that oxidative reactions may either mimic growth factor stimulation and stimulate cell proliferation or inhibit growth signals and activate PCD, in the same cell systems.

Topics: Animals; Calcium; Cell Death; Cell Division; Cell Line; Cell Survival; Humans; Mercuric Chloride; Naphthoquinones; Oxidants; PC12 Cells; Protein Kinase C; Signal Transduction; Sulfhydryl Compounds

In rat hepatocytes exposed to the quinones menadione and 2,3-dimethoxy-1,4-naphthoquinone (2,3-diOMe-1,4-NQ) a decrease in NAD+ is observed. DNA damage and activation of poly(ADP-ribose)polymerase are often associated with a decrease in NAD+. Using rat hepatocytes and human myeloid leukaemic cells (K562), we examined the extent of DNA damage induced by these quinones at non-toxic concentrations, i.e. at concentrations at which the cells completely exclude the dye trypan blue. Both quinones caused significant DNA damage at very low concentrations (5-100 microM). With 2,3-diOME-1,4-NQ (15 microM) or menadione (15 microM) single strand breaks (SSB) were observed at very early time points (less than 5 min), reaching a maximum between 20 and 30 min. Most SSB were repaired within 45 min of the removal of the quinones. Whilst extensive repair was observed within 4 hr of the removal of 2,3-diOMe-1,4-NQ (15 microM), only partial repair was observed following exposure to menadione (15 microM). SSB induced by 2,3-diOMe-1,4-NQ (15 microM) were completely inhibited by the iron chelator 1,10-phenanthroline (25 microM), whereas in cells exposed to menadione (15 microM) they were only partially inhibited. Finally, although the membrane integrity of K562 cells was unaffected by exposure to high concentrations of both quinones (less than or equal to 400 microM), cytostasis was observed at much lower concentrations (50 microM). Our results demonstrate that at very low concentrations these quinones induce extensive DNA damage possibly caused by hydroxyl radicals. The DNA damage was accompanied by an early cytostasis but no loss of membrane integrity.

Topics: Animals; Cell Division; DNA Damage; DNA, Single-Stranded; Dose-Response Relationship, Drug; Humans; Liver; NAD; Naphthoquinones; Phenanthrolines; Rats; Trypan Blue; Tumor Cells, Cultured; Vitamin K

The effects of redox cycling, alkylating and mixed redox cycling/alkylating benzo- and naphthoquinones were examined in electrically driven guinea pig left atria. Cardiac microsomal and mitochondrial NAD(P)H-dependent metabolism of the quinones and consequent generation of superoxide anion (O2.-) were also measured. Mixed redox cycling/alkylating 2-methyl-1,4-naphthoquinone, redox cycling 2,3-dimethoxy-1,4-naphthoquinone and alkylating p-benzoquinone determined concentration-dependent positive inotropic responses, whereas redox cycling 2,3,5,6-tetramethyl-p-benzoquinone had no effect. The positive inotropic effect of 2,3-dimethoxy-1,4-naphthoquinone was completely catecholamine-mediated, that of 2-methyl-1,4-naphthoquinone was approximately 70% adrenergic and 30% direct. p-Benzoquinone acted directly on heart muscle. In time, quinones with alkylating properties caused increases in the resting force of atria, whereas redox cycling quinones did not produce toxic effects. Mitochondrial NADH-oxidoreductase accounted for 90 to 95% of the metabolism of all quinones, whereas the contribution of the microsomal pathway was negligible. Considerable amounts of O2.- were produced by mitochondrial biotransformation of 2-methyl-1,4-naphthoquinone and 2,3-dimethoxy-1,4-naphthoquinone but not of 2,3,5,6-tetramethyl-p-benzoquinone and p-benzoquinone, suggesting a kind of relation between O2.- generation and the release of catecholamines.

Topics: Alkylating Agents; Animals; Atrial Function, Left; Benzoquinones; Guinea Pigs; Heart; Heart Atria; Mice; Microsomes; Mitochondria, Heart; Myocardial Contraction; NADH, NADPH Oxidoreductases; Naphthoquinones; Oxidation-Reduction; Quinones; Substrate Cycling; Superoxides; Vitamin K

In order to study oxidative stress in the lung, we have developed a rat lung slice model with compromised oxidative defences. Lung slices with markedly inhibited glutathione reductase activity (approximately 80% inhibition) were prepared by incubating slices, with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (100 microM) in an amino acid-rich medium for 45 min at 37 degrees. These lung slices had similar levels of GSH and ATP and polyamine uptake (a marker of alveolar epithelial type I and II cell function) to control rat lung slices. We have utilized these BCNU pretreated slices to study the effects of the herbicide, paraquat, in comparison to those of 2,3-dimethoxy-1,4-naphthoquinone, a potent redox cycler. Paraquat (10-100 microM) caused only minimal changes in the levels of GSH or ATP in control or compromised slices. In contrast, 2,3-dimethoxy-1,4-naphthoquinone caused a decrease in GSH in control slices but a markedly enhanced decrease in both GSH and ATP in compromised slices. Both compounds had only limited effects on putrescine and spermidine uptake in control slices. However, they caused a marked inhibition in compromised slices. Paraquat had little effect on 5-hydroxytryptamine uptake (a marker of endothelial cell function) in either control or compromised slices whereas the quinone inhibited uptake in the compromised slices. Thus, the lack of effect of paraquat on GSH and ATP does not support the involvement of oxidative stress in its toxicity. In contrast, using polyamine uptake, as a functional marker of alveolar epithelial cell damage, suggests a role for redox cycling. As paraquat is known to be accumulated primarily in alveolar type I and II cells (a small fraction of the lung cell population), our data suggest that only a small proportion of pulmonary GSH and ATP is present in alveolar epithelial type I and II cells but that much larger amounts may be present in endothelial cells. These studies highlight the problem of gross tissue measurements in heterogeneous tissues such as the lung.

Topics: Adenosine Triphosphate; Animals; Carmustine; Drug Synergism; Glutathione; Glutathione Reductase; Kinetics; Lung; Lung Diseases; Male; Naphthoquinones; Oxidation-Reduction; Paraquat; Putrescine; Rats; Rats, Inbred Strains; Serotonin; Spermidine

Exposure of hepatoma 1c1c7 cells to 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) resulted in a sustained elevation of cytosolic Ca2+, DNA single strand breaks and cell killing. DNA single strand break formation was prevented when cells were preloaded with either of the intracellular Ca2+ chelators, Quin 2 or BAPTA, to buffer the increase in cytosolic Ca2+ concentration induced by the quinone. DMNQ caused marked NAD+ depletion which was prevented when cells were preincubated with 3-aminobenzamide, an inhibitor of nuclear poly-(ADP-ribose)-synthetase activity, or with either of the two Ca2+ chelators. However, 3-aminobenzamide did not protect the hepatoma cells from loss of viability. Our results indicate that quinone-induced DNA damage, NAD+ depletion and cell killing are mediated by a sustained elevation of cytosolic Ca2+.

Topics: Animals; Calcium; Cell Survival; Chelating Agents; Cytosol; DNA Damage; Glutathione; Liver Neoplasms, Experimental; Mice; Naphthoquinones; Oxidation-Reduction; Quinones; Tumor Cells, Cultured