Mitochondria-targeted antioxidants do not prevent tumour necrosis factor-induced necrosis of L929 cells

Mitochondrial production of reactive oxygen species (ROS) is widely reported as a central effector during TNF-induced necrosis. The effect of a family of mitochondria-targeted antioxidants on TNF-induced necrosis of L929 cells was studied. While the commonly used lipid-soluble antioxidant BHA effectively protected cells from TNF-induced necrosis, the mitochondria-targeted antioxidants MitoQ₃, MitoQ₅, MitoQ₁₀ and MitoPBN had no effect on TNF-induced necrosis. Since BHA also acts as an uncoupler of mitochondrial membrane potential, two additional uncouplers were tested. FCCP and CCCP both provided dose-dependent inhibition of TNF-induced necrosis. In conclusion, the generation of mitochondrial ROS may not be necessary for TNF-induced necrosis. Instead, these results suggest alternative mitochondrial functions, such as a respiration-dependent process, are critical for necrotic death.     

Hydroxylamine acutely activates glucose uptake in L929 fibroblast cells

Nitroxyl (HNO) has a unique, but varied, set of biological properties including beneficial effects on cardiac contractility and stimulation of glucose uptake by GLUT1. These biological effects are largely initiated by HNO's reaction with cysteine residues of key proteins. The intracellular production of HNO has not yet been demonstrated, but the small molecule, hydroxylamine (HA), has been suggested as possible intracellular source. We examined the effects of this molecule on glucose uptake in L929 fibroblast cells. HA activates glucose uptake from 2 to 5-fold within two minutes. Prior treatment with thiol-active compounds, such as iodoacetamide (IA), cinnamaldehyde (CA), or phenylarsine oxide (PAO) blocks HA-activation of glucose uptake. Incubation of HA with the peroxidase inhibitor, sodium azide, also blocks the stimulatory effects of HA. This suggests that HA is oxidized to HNO by L929 fibroblast cells, which then reacts with cysteine residues to exert its stimulatory effects. The data suggest that GLUT1 is acutely activated in L929 cells by modification of cysteine residues, possibly the formation of a disulfide bond within GLUT1 itself.     

Methylene blue stimulates 2-deoxyglucose uptake in L929 fibroblast cells

Methylene blue (MB), a common cell stain, has been shown to inhibit nitric oxide synthase and guanylate cyclase, which has led to the recent use of MB in nitric oxide signaling studies. This study documents the effects of MB on 2-deoxyglucose (2DG) uptake in L929 fibroblast cells where uptake is controlled by a single glucose transporter, GLUT 1. MB significantly activates cytochalasin B-inhibitable glucose transport in a dose dependent fashion within 10 min. A maximal stimulation of up to 800% was achieved by 50 microM MB after a 45-min exposure. The Vmax of transport increased without a change in the Km, which was accomplished without a significant change in the GLUT 1 content. The reduced form of MB, did not stimulate 2DG uptake and potassium ferricyanide, an extracellular redox agent, prevented both the staining and stimulatory effects of MB suggesting MB is reduced at the cell surface before it enters L929 cells. Phenylarsine oxide did not block cell staining as noted in other cells lines, but it did inhibit both basal and MB-stimulated 2DG uptake. Likewise, methyl-beta-cyclodextrin, an agent used to remove membrane cholesterol, blocked both the staining and stimulatory effects of MB. The AMP analog, AICAR, inhibited rather than activated basal 2DG uptake, and it did not alter MB-stimulated uptake suggesting that AMP kinase activation is not critical to the MB effect. Wortmannin, an inhibitor of PI kinase, had no effect on MB-stimulated 2DG uptake. These data provide additional insight into the acute regulation of GLUT 1 transport activity in L929 cells.     

Bioenergetic aspects of apoptosis, necrosis and mitoptosis

In this review I summarize interrelations between bioenergetic processes and such programmed death phenomena as cell suicide (apoptosis and necrosis) and mitochondrial suicide (mitoptosis). The following conclusions are made. (I) ATP and rather often mitochondrial hyperpolarization (i.e. an increase in membrane potential, ΔΨ) are required for certain steps of apoptosis and necrosis. (II) Apoptosis, even if it is accompanied by ΔΨ and [ATP] increases at its early stage, finally results in a ΔΨ collapse and ATP decrease. (III) Moderate (about three-fold) lowering of [ATP] for short and long periods of time induces apoptosis and necrosis, respectively. In some types of apoptosis and necrosis, the cell death is mediated by a ΔΨ-dependent overproduction of ROS by the initial (Complex I) and the middle (Complex III) spans of the respiratory chain. ROS initiate mitoptosis which is postulated to rid the intracellular population of mitochondria from those that are ROS overproducing. Massive mitoptosis can result in cell death due to release to cytosol of the cell death proteins normally hidden in the mitochondrial intermembrane space.     

Nucleolus and large nucleolar aggregates of condensed chromatin in interphase nuclei of L 929 cells

Summary Three-dimensional reconstructions show that the nucleoli from L 929 cells are associated with one or several large aggregates of chromatin displaying a honeycomb-like structure. The form and the number of both nucleoli and honeycomb structures vary as the cells emerge from the resting state and enter exponential growth. Quantitative data show that the number of honeycomb structures decreases as the number of nucleoli diminishes; both numerical regressions are significant. In addition, the nucleoli and the honeycomb structures enlarge when the cells enter the exponential growth phase.In resting cells the number of honeycomb structures is correlated to the number of nucleoli. Therefore we conclude that the large nucleolar mass of condensed chromatin, which in L 929 cells displays a honeycomb structure, contains a portion of the nucleolar organizing region.     

Production of interleukin-1 and tumour necrosis factor in non-Hodgkin's lymphoma patients

Summary Peripheral blood monocytes from non-Hodgkin's lymphoma (NHL) patients were assessed for the monocyte functions with respect to their ability to secrete interleukin-1 and tumour necrosis factor (TNF) and their cytotoxic potential to tumour target WEHI 164 clone 13. Our results indicate comparable levels of interleukin-1 and TNF production by NHL patients. The cytotoxic potential by monocytes was also not depressed in these patients. The data obtained suggest normal monocyte functions in NHL patients.     

Induction of tumour necrosis factor by staphylococcal toxic shock toxin 1

Abstract Staphylococcal toxic shock syndrome toxin 1 (TSST1) induced the release of tumour necrosis factor (TNF) from human and rabbit monocytes in vitro. Nanogram amounts of TSST1 were sufficient to induce TNF release. There was considerable variation in response between cells from different rabbits and different donors. Rabbit monocytes were slightly more sensitive to TSST1 than were human monocytes. Release of TNF in vivo could explain many of the symptoms of toxic shock syndrome.     

Mechanism of teratogenesis: electron transfer, reactive oxygen species, and antioxidants

Teratogenesis has been a topic of increasing interest and concern in recent years, generating controversy in association with danger to humans and other living things. A veritable host of chemicals is known to be involved, encompassing a wide variety of classes, both organic and inorganic. Contact with these chemicals is virtually unavoidable due to contamination of air, water, ground, food, beverages, and household items, as well as exposure to medicinals. The resulting adverse effects on reproduction are numerous. There is uncertainty regarding the mode of action of these chemicals, although various theories have been advanced, e.g., disruption of the central nervous system (CNS), DNA attack, enzyme inhibition, interference with hormonal action, and insult to membranes, proteins, and mitochondria. This review provides extensive evidence for involvement of oxidative stress (OS) and electron transfer (ET) as a unifying theme. Successful application of the mechanistic approach is made to all of the main classes of toxins, in addition to large numbers of miscellaneous types. We believe it is not coincidental that the vast majority of these substances incorporate ET functionalities (quinone, metal complex, ArNO2, or conjugated iminium) either per se or in metabolites, potentially giving rise to reactive oxygen species (ROS) by redox cycling. Some categories, e.g., peroxides and radiation, appear to generate ROS by non-ET routes. Other mechanisms are briefly addressed; a multifaceted approach to mode of action appears to be the most logical. Our framework should increase understanding and contribute to preventative measures, such as use of antioxidants.     

14C]serine from phosphatidylserine labels ceramide and sphingomyelin in L929 cells: evidence for a new metabolic relationship between glycerophospholipids and sphingolipids

After incubation of L929 cells with [14C]serine and various effectors an inverse correlation between label in ceramide and phosphatidylserine (PS) was displayed. This surprising behavior of the two metabolites prompted us to check whether serine of PS could be a source for ceramide synthesis. We therefore incubated L929 cells for 30 min in serum-free medium with L-phosphatidyl-L-[3-14C]serine in the presence or in the absence of cycloserine, an established inhibitor of serine palmitoyltransferase. During this short period L-phosphatidyl-L-[3-14C]serine labeled ceramide and this label was suppressed by cycloserine. Then L929 cells were grown for 16-18 h in the presence of L-phosphatidyl-L-[3-14C]serine. After this period the label was seen in sphingomyelin. Labeling of ceramide and sphingomyelin by serine from PS provides evidence for a new metabolic relationship between glycerophospholipids and sphingolipids.     

Acute activation of glucose uptake by glucose deprivation in L929 fibroblast cells

Glucose is a very important energy source for a wide variety of cells, and the ability of cells to respond to changes in glucose availability or other cell stresses is of critical importance. Many mammalian cells respond to acute stress by increasing the V(max) of transport through GLUT1; the most ubiquitously expressed glucose transporter isoform. This study investigated the acute response of glucose uptake to glucose deprivation in L929 fibroblast cells--a cell line that expresses only the GLUT1 transporter. Results indicated that glucose deprivation of only a minute activated glucose uptake 10-fold and reached a maximum of 20-fold within 10 min. The activation was dose dependent and only partially muted by addition of up to 20mM pyruvate as an alternate energy source. In contrast to the kinetics of acute metabolic stress, glucose deprivation decreased the K(m) of transport, but did not alter the V(max). Maximal activation of glucose transport by glucose deprivation was completely additive to activation of transport by methylene blue--a stimulant that increased the V(max) of transport without a change in the K(m). Glucose-deprived activation of glucose transport was not inhibited by wortmannin or herbimycin A, but was completely inhibited by phenylarsine oxide. Altogether, the data indicate that L929 fibroblast cells respond quickly and robustly to the cell stress of glucose deprivation and methylene blue treatment by two distinct activation pathways.     

Priming effect of dextran sulphates on lipopolysaccharide-induced tumour necrosis factor production in mice

Abstract Dextran sulphate (DS) 500 (M.W. 500 000) is commonly used as a reticuloendothelial (RE) blocker. We found that lipopolysaccharide (LPS)-induced tumour necrosis factor (TNF) production in sera was enhanced when mice were pretreated with DS500. When mice were pretreated with DS1000 (M.W. 1 000 000), TNF activity in sera was also significantly enhanced by the LPS injection in comparison with the saline-treated group, but not by the pretreatment with the low molecular weight of DS5 (M.W. 5 000), neutral dextran (Dex) 500, or positively-charged diethylaminoethyl dextran (DEAE-Dex) 500. The enhancement of LPS-induced TNF production occurred from 2 h after DS500 pretreatment. Pretreatment with DS500 or DS1000 significantly suppressed the carbon clearance from the blood in mice from 2 h after DS injection, but this suppression was not detected by the pretreatment with DS5, Dex500, or DEAE-Dex500. We suggest that negative-charge and high molecular weight are essential for dextran derivatives to enhance LPS-induced TNF production, and that the enhancing effect of DS is closely related to the suppression of the RE function.     

Evaluation of anticancer effects of cerium oxide nanoparticles on mouse fibrosarcoma cell line

Cerium oxide nanoparticles are associated with anticancer effects. While protecting normal cells, these nanoparticles exert their anticancer effects via oxidative stress and apoptosis in the cancer cells. In this study, the anticancer properties of nanoceria on fibrosarcoma cell line are evaluated. Cerium oxide nanoparticles were synthesized by the coprecipitation method and their anticancer effects on mouse fibrosarcoma tumor cells (WEHI164) were investigated. Viability assay was evaluated by MTT, and the DC-FDA assay performed for the detection of reactive oxygen species. For apoptosis assay, the annexin V/PI test was done as well as measuring the mRNA and protein expression levels of Bax and Bcl2 by real-time PCR and western blot method, respectively. Characterization of nanoceria reveals that synthesized nanoceria has cubic floruit structure with a size of about 30 nm. Toxicity assessment results show that nanoceria increases ROS levels and induced apoptosis in a dose-dependent manner in cancer cells (WEHI164), whereas low levels of toxicity were observed in normal cells (L929), even at the concentrations above 250 µg/ml in MTT assay. Real-time PCR and western blot assays showed that nanoceria could significantly increase the Bax expression in cancer cells. The results showed that nanoceria could act as a potential therapeutic agent for the treatment of fibrosarcoma.     

Influence of antioxidants on NO-dependent induction of heme oxygenase-1 in U937 monocytes

Thiol antioxidants are known to inhibit the nitric oxide-dependent induction of the hemoxygenase-1 gene (HOX-1). To estimate the degree to which the inhibitory effect of thiol antioxidants is accounted for by them scavenging oxidized NO derivatives or their precursors, the reactive oxygen and nitrogen species (ROS and RNS), we studied the inhibitory effect of nonthiol antioxidants: dimethyl sulfoxide, dimethylthiourea, sodium salicylate, sodium formate, uric acid, catalase, and superoxide dismutase. Partial inhibition of NO-dependent HOX-1 induction was observed in the presence of the nonpolar HO scavengers dimethyl sulfoxide and dimethylthiourea. The antioxidants which selectively bind other ROS had no effect on HOX-1 expression. To reveal the role of RNS in NO-dependent HOX-1 induction, cells were treated with the NO-generating compound DPTA-NO in the presence of 2-phenyl-4,4,5,5,-tetramethylimidazole-1-oxyl 3 oxide (PTIO), which oxidizes NO to NO₂. PTIO proved to significantly enhance NO-dependent HOX-1 induction. Thiol antioxidants completely inhibited the stimulating effect of PTIO, which is evidence that their inhibitory effect is explained by RNS scavenging. The results of this study indicate that antioxidants can be used to modulate the cell response to NO.Translated from Molekulyarnaya Biologiya, Vol. 39, No. 1, 2005, pp. 89–95.Original Russian Text Copyright © 2005 by Litvinov, Prasolov, Bouton, Drapier, Turpaev.     

Mitochondrial metabolism of reactive oxygen species

Oxidative stress is considered a major contributor to etiology of both normal senescence and severe pathologies with serious public health implications. Mitochondria generate reactive oxygen species (ROS) that are thought to augment intracellular oxidative stress. Mitochondria possess at least nine known sites that are capable of generating superoxide anion, a progenitor ROS. Mitochondria also possess numerous ROS defense systems that are much less studied. Studies of the last three decades shed light on many important mechanistic details of mitochondrial ROS production, but the bigger picture remains obscure. This review summarizes the current knowledge about major components involved in mitochondrial ROS metabolism and factors that regulate ROS generation and removal. An integrative, systemic approach is applied to analysis of mitochondrial ROS metabolism, which is now dissected into mitochondrial ROS production, mitochondrial ROS removal, and mitochondrial ROS emission. It is suggested that mitochondria augment intracellular oxidative stress due primarily to failure of their ROS removal systems, whereas the role of mitochondrial ROS emission is yet to be determined and a net increase in mitochondrial ROS production in situ remains to be demonstrated.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 246–264.Original Russian Text Copyright © 2005 by Andreyev, Kushnareva, Starkov.This revised version was published online in April 2005 with corrections to the post codes.     

Tumour-cell-induced production of tumour necrosis factor by monocytes of gastric cancer patients receiving BCG immunotherapy

Summary Human peripheral blood monocytes cocultured with tumour cells were used as an in vitro model of in situ interactions between tumour-infiltrating macrophages and the tumour. Tumour cells stimulated de novo expression of the human tumour necrosis factor (TNF) gene in monocytes and caused the release of TNF into the culture supernatant. A group of 14 patients with stage IVA gastric cancer receiving adjuvant chemotherapy (5-FU, Adriamycin, mitomycin C: FAM) or immunochemotherapy (BCG+FAM) was investigated for the ability of monocytes to produce TNF in vitro upon stimulation with tumour cells or purified protein derivative of tuberculin (PPD). Patients were followed at biweekly intervals, i.e. before each instillation of BCG epicutaneously over a period of 10 weeks. It was found that monocytes of some patients receiving BCG at the end of the observation period had an enhanced ability to produce TNF following stimulation with tumour cells. In contrast, such production was not substantially altered during the study period in patients on chemotherapy. PPD-induced TNF production was much weaker and was not significantly changed during this observation time. We infer that BCG immunotherapy may induce the subtle changes in some cancer patients that lead to an increased interaction between monocytes and tumour cells and result in enhanced production of cytokine(s) with antitumour properties.     

Cathepsin L deficiency results in reactive oxygen species (ROS) accumulation and vascular cells activation

Recent evidence suggests a link between cathepsin L (CTSL) and vascular diseases. However, its contribution to reactive oxygen species (ROS) homeostasis in the vasculature remains unknown. p66shc is a redox enzyme implicated in mitochondrial ROS generation and translation of oxidative signals. In this study, we explored the relationship between CTSL and oxidative damage in vasculature and whether the oxidative damage is mediated by p66shc.Carotid arteries from aged mice (24 months old) showed a reduction in CTSL expression compared with young wild-type mice (4 months old). Local knockdown of CTSL in carotid arteries of young mice by adenoviral vector encoding the short hairpin RNA targeting CTSL leading to premature vascular aging, as shown by mitochondrial disruption, increased β-galactosidase–positive cells, reduced telomerase activity, and up-regulation of p66shc. Knockdown of CTSL decreased the expression of mitochondrial oxidative phosphorylation (OXPHOS) complexes I, III, and IV, leading to increased mitochondrial ROS and hyperpolarization of the mitochondrial membrane in vitro. Furthermore, knockdown of CTSL also stimulated ROS production and senescence in vascular cells, accompanied by the up-regulation of p66shc.However, p66shc knockdown blunted the alteration in ROS production, and senescence in CTSL knockdown vascular cells. This study suggests that CTSL knockdown partially induces vascular cells damage via increased ROS production and up-regulation of p66shc.     

Oestrogen-mediated suppression of tumour necrosis factor alpha-induced apoptosis in MCF-7 cells: subversion of Bcl-2 by anti-oestrogens

In oestrogen receptor (ER)-positive breast carcinoma cells, 17β-oestradiol suppresses a dose-dependent induction of cell death by tumour necrosis factor alpha (TNF). The ability of oestrogens to promote cell survival in ER-positive breast carcinoma cells is linked to a coordinate increase in Bcl-2 expression, an effect that is blocked with the pure anti-oestrogen ICI 182,780. The role of Bcl-2 in MCF-7 cell survival was confirmed by stable overexpression of Bcl-2 which resulted in suppression of apoptosis induced by doxorubicin (DOX), paclitaxel (TAX) and TNF as compared to vector-control cells. The pure anti-oestrogen ICI 182,780 in combination with TNF, DOX or TAX potentiated apoptosis in vector-transfected cells. Interestingly, pre-treatment with ICI 182,780 markedly enhanced chemotherapeutic drug- or TNF-induced apoptosis in Bcl-2 expressing cells, an effect that was correlated with ICI 182,780 induced activation of c-Jun N-terminal kinase. Our results suggest that the effects of oestrogens/anti-oestrogens on the regulation of apoptosis may involve coordinate activation of signalling events and Bcl-2 expression.     

Traditional reactive carbonyl scavengers do not prevent the carbonylation of brain proteins induced by acute glutathione depletion

Abstract

This study investigated the effect of reactive carbonyl species (RCS)-trapping agents on the formation of protein carbonyls during depletion of brain glutathione (GSH). To this end, rat brain slices were incubated with the GSH-depletor diethyl maleate in the absence or presence of chemically different RCS scavengers (hydralazine, methoxylamine, aminoguanidine, pyridoxamine, carnosine, taurine and z-histidine hydrazide). Despite their strong reactivity towards the most common RCS, none of the scavengers tested, with the exception of hydralazine, prevented protein carbonylation. These findings suggest that the majority of protein-associated carbonyl groups in this oxidative stress paradigm do not derive from stable lipid peroxidation products like malondialdehyde (MDA), acrolein and 4-hydroxynonenal (4-HNE). This conclusion was confirmed by the observation that the amount of MDA-, acrolein- and 4-HNE-protein adducts does not increase upon GSH depletion. Additional studies revealed that the efficacy of hydralazine at preventing carbonylation was due to its ability to reduce oxidative stress, most likely by inhibiting mitochondrial production of superoxide and/or by scavenging lipid free radicals.     

Phytotoxic potential of Senna occidentalis (L.) Link extracts on seed germination and oxidative stress of Ipê seedlings

• Senna occidentalis is an invasive plant producing a series of allelochemicals that might inhibit the development of other plants. The objective of this study was to assess the phytotoxic effect of S. occidentalis extracts on the germination, development and antioxidant defence of the native species Tabebuia chrysotricha, T. pentaphylla, T. roseoalba and Handroanthus impetiginosus (Ipê species).
• We evaluated the effects of chemicals extracted from S. occidentalis on the germination rate, germination speed index (GSI) and biometric parameters of the test species under controlled conditions. The effect of the extracts on the pigment content, amount of H₂O₂ and malondialdehyde (MDA), and the activity of the antioxidant enzymes in roots and leaves were also tested.
• Alkaloids, coumarins, phenols, saponins, free steroids and condensed tannins were present in all extracts of S. occidentalis, while catechins were present only in leaf and stem extracts. Stem and root extracts caused a growth reduction in all Ipê species and total inhibition of seed germination in T. chrysotricha and T. roseoalba. All target species showed an increase in H₂O₂ and MDA in radicles and leaves. Oxidative stress contributed strongly to the morphological changes, such as seed blackening, thinning and darkening of radicle tips and reduction of biomass allocation in all Ipê species.
• Although there was activation of antioxidant defence mechanisms, such as an increase in activity of ascorbate peroxidase (APX) and peroxidase (POD) enzymes, the joint action of the allelochemicals caused phytotoxicity, leading to cell dysfunction in all Ipê species.