Aberrant nuclear morphology in the mouse myeloma SP2/0 cell line

The mouse myeloma SP2/0 cell line when grown in supplemented Dulbecco's modified Eagle's media spontaneously produced aberrant nucleated cells which increased in frequency with cell culture age. These cells underwent cytological changes associated with apoptosis, that is, the condensation of chromatin followed by karyorrhexis and the production of small apoptotic bodies. Aberrant cells were induced by media changes, centrifugation, and temperature shocking. The rapid induction of aberrant cells by a media change suggests that the mechanism of fragmentation was not associated with cell division.     

Roles of reactive oxygen species and antioxidants in ovarian toxicity

Proper functioning of the ovary is critical to maintain fertility and overall health, and ovarian function depends on the maintenance and normal development of ovarian follicles. This review presents evidence about the potential impact of oxidative stress on the well-being of primordial, growing and preovulatory follicles, as well as oocytes and early embryos, examining cell types and molecular targets. Limited data from genetically modified mouse models suggest that several antioxidant enzymes that protect cells from reactive oxygen species (ROS) may play important roles in follicular development and/or survival. Exposures to agents known to cause oxidative stress, such as gamma irradiation, chemotherapeutic drugs, or polycyclic aromatic hydrocarbons, induce rapid primordial follicle loss; however, the mechanistic role of ROS has received limited attention. In contrast, ROS may play an important role in the initiation of apoptosis in antral follicles. Depletion of glutathione leads to atresia of antral follicles in vivo and apoptosis of granulosa cells in cultured antral follicles. Chemicals, such as cyclophosphamide, dimethylbenzanthracene, and methoxychlor, increase proapoptotic signals, preceded by increased ROS and signs of oxidative stress, and cotreatment with antioxidants is protective. In oocytes, glutathione levels change rapidly during progression of meiosis and early embryonic development, and high oocyte glutathione at the time of fertilization is required for male pronucleus formation and for embryonic development to the blastocyst stage. Because current evidence suggests that oxidative stress can have significant negative impacts on female fertility and gamete health, dietary or pharmacological intervention may prove to be effective strategies to protect female fertility.     

PARP-1 inhibition induces a late increase in the level of reactive oxygen species in cells after ionizing radiation

Poly(ADP-ribose) polymerase 1 (PARP1), an enzyme activated by DNA strand breaks, synthesizes polymers of poly(ADP-ribose) (PAR) that modify chromatin and other proteins and play a role in DNA repair. Inhibition of PARP1 activity is considered a potentially important strategy in clinical practice, especially to sensitize tumor cells to chemo- and radio-therapy. Here we examined the influence of inhibition of PARP1 on formation of reactive oxygen species (ROS) and on DNA repair in cells exposed to ionizing radiation (IR). K562 (human myelogenous leukaemia) cells were grown and exposed to 4 or 12Gy of ionizing radiation in presence or absence of the PARP inhibitor NU1025 (100μM). Intracellular ROS were assayed using the probe 2,7-dichlorofluorescein with detection by flow cytometry and the rejoining of DNA strand breaks were followed by alkaline single cell gel electrophoresis (comet) assays. In untreated cells a significant increase in PAR formation occurred during the first 5min after IR, followed by a gradual decrease up to 30min. Addition of a PARP inhibitor arrested the production of PAR almost completely and decreased the rate of rejoining of DNA strand breaks significantly; however, 3h after irradiation we observed no difference in the amount of DNA strand breaks between PARP inhibitor-treated and untreated cells. Twelve to 48h after irradiation, an increase of ROS concentration was observed in irradiated cells and ROS levels in PARP inhibitor-treated cells were significantly higher than in cells without inhibitor. Irradiated cells grown in the presence or absence of PARP inhibitor did not differ in the frequencies of apoptotic and necrotic cells or in the activity of caspases at 24, 48 and 72h after irradiation. Poly(ADP-ribosylation) and inhibition of PARP1 appeared to modulate DNA strand break rejoining and influence the concentration of ROS in irradiated cells.     

Role of mitochondrial reactive oxygen species in hypoxia-dependent increase in intracellular calcium in pulmonary artery myocytes

Previous studies examining the role of mitochondria-derived reactive oxygen species (ROS) in hypoxic responses have been mainly conducted in isolated lungs and cultured pulmonary artery smooth muscle cells (PASMCs) using mitochondrial inhibitors, and yielded largely conflicting results. Here we report that in freshly isolated mouse PASMCs, which are devoid of the mixed responses from multi-types of cells in lungs and significant changes in gene expression in cultured cells, the mitochondrial electron transport chain (ETC) complex I, II, or III inhibitors blocked hypoxia-induced increases in intracellular ROS and Ca2+ concentration ([ROS]i and [Ca2+]i) without effects on their resting levels. Inhibition of the complex I plus II and/or III did not produce an additive effect. Glutathione peroxidase-1 (Gpx1) or catalase gene overexpression to enhance H2O2 removal remarkably reduced hypoxic increases in [ROS]i and [Ca2+]i, whereas Gpx1 gene deletion had the opposite effect. None of these genetic modifications changed the resting [ROS]i and [Ca2+]i. H2O2 at 51 microM caused a similar increase in DCF fluorescence ([ROS]i) as that by hypoxia, but only induced 33% of hypoxic increase in [Ca2+]i. Moreover, H2O2 (5.1 microM) reversed the inhibition of the hypoxia-induced increase in [Ca2+]i by rotenone. Collectively, our study using various mitochondrial inhibitors and genetic approaches demonstrates that in response to acute hypoxia, the mitochondrial ETC molecules prior to the complex III ubisemiquinone site act as a functional unit to increase the generation of ROS, particularly H2O2, which is important for, but may not fully cause, the hypoxic increase in [Ca2+]i in freshly isolated PASMCs.     

A review of the interaction among dietary antioxidants and reactive oxygen species

During normal cellular activities, various processes inside of cells produce reactive oxygen species (ROS). Some of the most common ROS are hydrogen peroxide (H(2)O(2)), superoxide ion (O(2)(-)), and hydroxide radical (OH(-)). These compounds, when present in a high enough concentration, can damage cellular proteins and lipids or form DNA adducts that may promote carcinogenic activity. The purpose of antioxidants in a physiological setting is to prevent ROS concentrations from reaching a high-enough level within a cell that damage may occur. Cellular antioxidants may be enzymatic (catalase, glutathione peroxidase, superoxide dismutase) or nonenzymatic (glutathione, thiols, some vitamins and metals, or phytochemicals such as isoflavones, polyphenols, and flavanoids). Reactive oxygen species are a potential double-edged sword in disease prevention and promotion. Whereas generation of ROS once was viewed as detrimental to the overall health of the organism, advances in research have shown that ROS play crucial roles in normal physiological processes including response to growth factors, the immune response, and apoptotic elimination of damaged cells. Notwithstanding these beneficial functions, aberrant production or regulation of ROS activity has been demonstrated to contribute to the development of some prevalent diseases and conditions, including cancer and cardiovascular disease (CVD). The topic of antioxidant usage and ROS is currently receiving much attention because of studies linking the use of some antioxidants with increased mortality in primarily higher-risk populations and the lack of strong efficacy data for protection against cancer and heart disease, at least in populations with adequate baseline dietary consumption. In normal physiological processes, antioxidants effect signal transduction and regulation of proliferation and the immune response. Reactive oxygen species have been linked to cancer and CVD, and antioxidants have been considered promising therapy for prevention and treatment of these diseases, especially given the tantalizing links observed between diets high in fruits and vegetables (and presumably antioxidants) and decreased risks for cancer.     

Effects of proximate cholesterol precursors and steroid hormones on mouse myeloma growth in serum-free medium

Summary The proximate cholesterol precursors lathosterol, 7-dehydrocholesterol and desmosterol supported the growth of NS-1 and X63 mouse myeloma cells. These cells and X63.653 cells are cholesterol auxotrophs, yet each was able to convert [³H]lathosterol to [³H]cholesterol. These results are consistent with the conclusion that cholesterol auxotrophy in these myeloma cells is due to a deficiency in 3-ketosteroid reductase activity. The steroid hormones testosterone, progesserone and hydrocortisone could not replace cholesterol as a medium supplement. These results provide a greater understanding of the cholesterol auxotrophy characteristic of cell lines clonally-derived from the MOPC 21 myeloma tumor, and they provide a rational basis for the use of sterols in defined culture medium for mouse myeloma cells. This work was supported by National Institute of Health grants CA40294 and CA37589 to G. H. Sato and by a grant from RJR nabisco Inc. Editor's Statement These results help identify the defect in myeloma cells leading to cholesterol auxotrophy. The use of these cells in hybridoma derivation adds practical utility to a detailed appreciation of cholesterol metabolism in these cultures.     

Externally applied pressure activates pancreatic stellate cells through the generation of intracellular reactive oxygen species

Local tissue pressure is higher in chronic pancreatitis than in the normal pancreas. We reported recently that pressure application induces synthesis of extracellular matrix (ECM) and cytokines in pancreatic stellate cells (PSCs) and that epigallocatechin gallate (EGCG), a potent antioxidant, inhibits the transformation of PSCs from quiescent to activated phenotype and ethanol-induced synthesis of ECM and cytokines in PSCs. These results suggest that oxidative stress and reactive oxygen species (ROS) are important in PSC activation. The aim of this study was to clarify the effects of ROS on activation and functions of pressure-stimulated PSCs. We used freshly isolated rat PSCs and culture-activated PSCs. Pressure was applied on rat cultured PSCs by adding compressed helium gas into a pressure-loading apparatus. PSCs were cultured with or without antioxidants (EGCG and N-acetyl cysteine) under normal or elevated pressure. Externally applied high pressure (80 mmHg) resulted in a gradual decrease of superoxide dismutase activity in PSCs and increased intracellular ROS generation as early as 30 s, reaching a peak level at 1 h. Antioxidants significantly inhibited ROS generation. Pressure increased the expression levels of alpha-smooth muscle actin, alpha(1)(I)-procollagen, and TGF-beta1 in PSCs. EGCG suppressed these alterations, abolished pressure-induced phosphorylation of p38 MAPK, and suppressed pressure-induced PSC transformation to activated phenotype. Our results indicated that ROS is a key player in pressure-induced PSC activation and ECM synthesis. Antioxidants could be potentially effective against the development of pancreatic fibrosis in patients with chronic pancreatitis.     

Hoechst 33342 induces radiosensitization in malignant glioma cells via increase in mitochondrial reactive oxygen species

Abstract

Mitochondrial DNA plays an important role in cellular sensitivity to cancer therapeutic agents. Hoechst 33342, a DNA minor groove binding ligand, has shown radiosensitizing effects in different cancer cell lines. In the present study, the possible binding of Hoechst 33342 with mitochondrial DNA, isolated from human cerebral glioma (BMG-1) cells, was investigated and consequences of this binding on excessive reactive oxygen species (ROS) generation in irradiated BMG-1 cells were studied. Alteration in the fluorescence spectroscopic characteristics of Hoechst 33342 suggested binding of Hoechst 33342 with isolated mitochondria and mitochondrial DNA. Persistent increase in level of ROS in the presence of Hoechst 33342 has been observed, which was further enhanced in irradiated cells. Investigations using inhibitors of ETC complex I suggested that mitochondrial bound Hoechst 33342 contributed to increased ROS, which was associated with alteration in ΔΨm and antioxidant machinery. These factors appeared to contribute in potentiating radiation-induced cell death in BMG-1 cells. The finding from these studies will be useful in designing better anti-cancer strategies.     

Primitive erythropoiesis of mouse teratocarcinoma stem cells PCC3/A/1 in serum-free medium

Mouse teratocarcinoma stem cells PCC3/A/1 differentiated into various types of cells, such as red cells, when they were grown in serum-free medium containing transferrin and bovine serum albumin on a KCF cell feeder layer. These red cells were stained well with 2,7-diaminofluorene (DAF), and therefore were erythroid cells. They were nucleated and contained embryonic globin chains, immunologically identified with antiembryonic hemoglobin antisera after acid urea Triton X-100 polyacrylamide gel electrophoresis (UT-PAGE). The addition of erythropoietin to the culture medium enhanced the production of both embryonic and adult globin chains. The addition of interleukin-3 also enhanced the production of embryonic globin chains, but not the production of adult globin chains. These results indicated that primitive erythropoiesis of PCC3/A/1 teratocarcinoma cells did not require exogenous addition of any hematopoietic factor such as erythropoietin or interleukin-3. This culture system will be a new model system for investigating the factors regulating the primitive erythropoiesis in yolk sac blood islands.     

Characterization and serial propagation of mouse prostate epithelial cells in serum-free medium

Epithelial cell cultures derived from the ventral prostate of normal adult mice have been propagated in serum-free medium. The cultures were initiated and maintained in Ham's F-12 nutrient mixture supplemented with insulin (5 micrograms/ml), EGF (10 ng/ml), hydrocortisone (0.5 micrograms/ml), cholera toxin (10 ng/ml), bovine pituitary extract (25 micrograms protein/ml) and antibiotics. The cells exhibited microvilli on cell surfaces, interdigitations and junctional complexes including desmosomes between cells, and cytokeratins in cytoplasm which are characteristic of epithelial cells. In addition, the cells exhibited the tissue-specific markers, prostatic acid phosphatase and prostate-specific antigen.     

Expression and purification of a recombinant human glycoprotein in mouse hybridoma SP2/0-AG14 cells

The cDNA for human erythropoietin (hEPO) inserted into the mammalian expression vector BMCGSNeo was introduced into SP2/0-Ag14 cells and a transformant producing large amounts of hEPO was established. The recombinant hEPO in conditioned medium was purified by immunoaffinity and gel filtration column chromatographies. The purified hEPO had full in vitro biological activity, but low in vivo biological activity.     

Effect of quercetin and its conjugated metabolite on the hydrogen peroxide-induced intracellular production of reactive oxygen species in mouse fibroblasts

To clarify the antioxidative role of quercetin metabolites in cellular oxidative stress, we measured the inhibitory effects of the quercetin aglycon and quercetin 3-O-beta-D-glucuronide (Q3GA), which is one of the quercetin metabolites in the blood after an intake of quercetin-rich food, on the production of hydrogen peroxide (H2O2)-induced intracellular reactive oxygen species in mouse fibroblast 3T3 cultured cells. When the cells were exposed to H2O2 in the presence of quercetin or Q3GA, Q3GA was found to be less effective than quercetin. In the case of a pretreatment with quercetin or Q3GA before the exposure, Q3GA, but not the quercetin aglycon, exerted an inhibitory effect, although its cellular uptake was unlikely. The quercetin aglycon appeared to fail in its antioxidative effect due to metabolic conversion into isorhamnetin conjugates, with substantial oxidative degradation resulting from the pretreatment. It is, therefore, suggested that quercetin metabolites take part in the protection of intracellular oxidative stress induced by the extraneous attack of H2O2.     

Relationship between the action of reactive oxygen and nitrogen species on bilayer membranes and antioxidants

Membrane lipid peroxidation (LPO) induced by hydroxyl (*OH) and ascorbyl (*Asc) radicals and by peroxynitrite (ONOO-) was investigated in asolectin (ASO), egg phosphatidylcholine (PC) and PC/phosphatidic acid mixtures (PC:PA) liposomes and rat liver microsomes (MC). Enthalpy variation (DeltaH) of PC:PA at different molar ratios were obtained by differential scanning calorimetry. It was also evaluated the LPO inhibition by quercetin, melatonin and Vitamin B6. The oxidant effect power follows the order *OH approximately *Asc > ONOO- on PC and MC; whilst on ASO liposomes, it follows *Asc > *OH approximately ONOO-. Increasing amounts of PA in PC liposomes resulted in lower levels of LPO. The DeltaH values indicate a more ordered membrane arrangement as a function of PA amount. The results were discussed in order to provide a complete view involving the influence of membranes, oxidants and antioxidants intrinsic behavior on the LPO dynamics.     

Aggrecan degradation in chondrocytes is mediated by reactive oxygen species and protected by antioxidants

Reactive oxygen species (ROS) are implicated in aging of cartilage and in the pathogenesis of osteoarthritis. However, the biological role of chondrocytes-derived ROS has not been elucidated. An in-vitro model was developed to study the role of chondrocyte-derived ROS in cartilage matrix degradation. The primary articular chondrocytes were cultured and the aggrecan matrix was radiolabeled with 35-sulfate. The labeled aggrecan matrix was washed to remove unincorporated label and chondrocytes were returned to serum free balanced salt solution. The cell-monolayer-matrix sensitivity to oxidative damage due to either hydrogen peroxide or glucose oxidase was established by monitoring the release of labeled aggrecan into the medium. Lipopolysaccharide (LPS) treatment of chondrocytemonolayer enhanced the release of labeled aggrecan. Catalase significantly prevented the release of labeled aggrecan in LPS-chondrocyte cultures, suggesting a role for chondrocyte-derived hydrogen peroxide in aggrecan degradation. Superoxide dismutase or boiled catalase had no such inhibitory effect. The effect of several antioxidants on LPS-chondrocyte-dependent aggrecan degradation was examined. Hydroxyl radical scavengers (mannitol and thiourea) significantly decreased aggrecan degradation. A spin trapping agent N-tert-butyl-phenylnitrone (but not its inactive analog tert-butyl-phenylcarbonate) significantly decreased aggrecan degradation. Butylated hydroxytoluene also inhibited aggrecan degradation, whereas the other lipophilic antioxidant tested, propyl gallate, had a marked dose-dependent inhibitory effect. These data indicate that general antioxidants, hydroxyl radical scavengers, antioxidant vitamins, iron chelating agents, lipophilic antioxidants, and spin trapping agents can influence chondrocyte-dependent aggrecan degradation. These studies support the role of a chondrocyte-dependent oxidative mechanism in aggrecan degradation and indicate that antioxidants can prevent matrix degradation and therefore may have a preventive or therapeutic value in arthritis. The enhancement of oxidative activity in chondrocytes and its damaging effect on matrix may be an important mechanism of matrix degradation in osteoarthritis.     

Aggrecan degradation in chondrocytes is mediated by reactive oxygen species and protected by antioxidants

Reactive oxygen species (ROS) are implicated in aging of cartilage and in the pathogenesis of osteoarthritis. However, the biological role of chondrocytes-derived ROS has not been elucidated. An in-vitro model was developed to study the role of chondrocyte-derived ROS in cartilage matrix degradation. The primary articular chondrocytes were cultured and the aggrecan matrix was radiolabeled with 35-sulfate. The labeled aggrecan matrix was washed to remove unincorporated label and chondrocytes were returned to serum free balanced salt solution. The cell-monolayer-matrix sensitivity to oxidative damage due to either hydrogen peroxide or glucose oxidase was established by monitoring the release of labeled aggrecan into the medium. Lipopolysaccharide (LPS) treatment of chondrocytemonolayer enhanced the release of labeled aggrecan. Catalase significantly prevented the release of labeled aggrecan in LPS-chondrocyte cultures, suggesting a role for chondrocyte-derived hydrogen peroxide in aggrecan degradation. Superoxide dismutase or boiled catalase had no such inhibitory effect. The effect of several antioxidants on LPS-chondrocyte-dependent aggrecan degradation was examined. Hydroxyl radical scavengers (mannitol and thiourea) significantly decreased aggrecan degradation. A spin trapping agent N-tert-butyl-phenylnitrone (but not its inactive analog tert-butyl-phenylcarbonate) significantly decreased aggrecan degradation. Butylated hydroxytoluene also inhibited aggrecan degradation, whereas the other lipophilic antioxidant tested, propyl gallate, had a marked dose-dependent inhibitory effect. These data indicate that general antioxidants, hydroxyl radical scavengers, antioxidant vitamins, iron chelating agents, lipophilic antioxidants, and spin trapping agents can influence chondrocyte-dependent aggrecan degradation. These studies support the role of a chondrocyte-dependent oxidative mechanism in aggrecan degradation and indicate that antioxidants can prevent matrix degradation and therefore may have a preventive or therapeutic value in arthritis. The enhancement of oxidative activity in chondrocytes and its damaging effect on matrix may be an important mechanism of matrix degradation in osteoarthritis.     

Reactive oxygen species modulates the intracellular level of HBx viral oncoprotein

HBx (hepatitis B virus X) viral oncoprotein is a multifunctional protein of which the cellular level may be one of the important factors in determining HBV-mediated pathological progression of liver diseases, chronic hepatitis, and hepatocellular carcinoma. Our previous work revealed that adriamycin, a chemotherapeutic agent, caused a marked increase in the intracellular level of HBx by retarding its rapid degradation. In the present study, modulation of HBx expression was found to be confined to adriamycin but not to other chemotherapeutic agents, cisplatin and 5-fluorouracil. Interestingly, adriamycin caused a rapid increase of reactive oxygen species (ROS) and its accumulation continued until 24h. In contrast, two other agents had little effect on ROS generation, suggesting the possible involvement of ROS in the HBx regulation. In fact, direct addition of H(2)O(2) to the cells significantly increased the level of HBx protein in HBx-expressing ChangX-34 cells as well as in hepatitis B virus-related hepatoma cells, PLC/PRF/5 and HepG2.2.15 cells. Furthermore, antioxidants, N-acetyl-cysteine and pyrrolidinedithiocarbamate (PDTC), completely abolished the increase of HBx protein induced by adriamycin, indicating that adriamycin modulates the intracellular HBx level via ROS generation. Together, these findings provide a novel aspect of HBx regulation by cellular ROS level. Therefore, intracellular microenvironments generating ROS such as severe inflammation may aggravate the pathogenesis of liver disease by accumulating the HBx level.     

Effects of reactive oxygen species and interplay of antioxidants during physical exercise in skeletal muscles

A large number of researches have led to a substantial growth of knowledge about exercise and oxidative stress. Initial investigations reported that physical exercise generates free radical-mediated damages to cells; however, in recent years, studies have shown that regular exercise can upregulate endogenous antioxidants and reduce oxidative damage. Yet, strenuous exercise perturbs the antioxidant system by increasing the reactive oxygen species (ROS) content. These alterations in the cellular environment seem to occur in an exercise type-dependent manner. The source of ROS generation during exercise is debatable, but now it is well established that both contracting and relaxing skeletal muscles generate reactive oxygen species and reactive nitrogen species. In particular, exercises of higher intensity and longer duration can cause oxidative damage to lipids, proteins, and nucleotides in myocytes. In this review, we summarize the ROS effects and interplay of antioxidants in skeletal muscle during physical exercise. Additionally, we discuss how ROS-mediated signaling influences physical exercise in antioxidant system.     

The level of DNA damage in mouse hematopoietic cells and in frog and human blood cells,as induced by the action of reactive oxygen species in vitro

Comparative studies of the level of DNA damage induced in vitro by X-rays (0–8 Gy) or hydrogen peroxide (0–300 µM) in cells of blood, spleen, and bone marrow of mice and in blood cells of frogs and humans were performed using the alkaline comet assay. For both agents, the levels of induced DNA damage in leucocytes/splenocytes of mice were higher than those in blood cells of frogs and humans, while in human leucocytes, they were comparable with those in frog blood cells. The rate of DNA repair in frog blood cells was very slow. The results suggest that the levels of radiation-induced DNA damage are not in accordance with species radiosensitivity (according to LD_50/30) but rather with the intrinsic peculiarities of cells.     

Rodent and human mast cells produce functionally significant intracellular reactive oxygen species but not nitric oxide

In immunity, reactive oxygen species (ROS) and nitric oxide (NO) are important antimicrobial agents and regulators of cell signaling and activation pathways. However, the cellular sources of ROS and NO are much debated. Particularly, there is contention over whether mast cells, key secretory cells in allergy and immunity, can generate these chemical species, and if so, whether they are of functional significance. We therefore examined directly by flow cytometry the capacity of mast cells to generate intracellular ROS and NO using the respective cell-permeable fluorescent probes dichlorodihydrofluorescein and diaminofluorescein and evaluated the effects of inhibitors of ROS and NO synthesis on cell degranulation. For each of three mast cell types (rat peritoneal mast cells, mouse bone marrow-derived mast cells, and human blood-derived mast cells), degranulation stimulated by IgE/antigen was accompanied by production of intracellular ROS but not NO. Inhibition of ROS production led to reduced degranulation, indicating a facilitatory role for ROS, whereas NO synthase inhibitors were without effect. Likewise, bacterial lipopolysaccharide and interferon-gamma over a wide range of conditions failed to generate intracellular NO in mast cells, whereas these agents readily induced intracellular NO in macrophages. NO synthase protein, as assessed by Western blotting, was readily induced in macrophages but not mast cells. We conclude that rodent and human mast cells generate intracellular ROS but not NO and that intracellular ROS but not intracellular NO are functionally linked to mast cell degranulation.