Induced Reactive Oxygen Species Formation Research Articles

Estradiol-17β increases 12- and 15-lipoxygenase (type2) expression and activity and reactive oxygen species in human umbilical vascular smooth muscle cells

The net vascular effect of estrogens on the vasculature is still under debate. Here we tested the effects of estradiol- 17β (E2) as well as estrogen-receptor subtype specific and non-specific agonists and antagonists on the expression and eicosanoid production of lipoxygenase (LO) enzymes expressed in culture human umbilical vascular smooth muscle cells (VSMC), the platelet type 12LO and 15LO type 2. E2 increased 12 and 15LO mRNA expression by 2–3 folds and elicited an acute 50% increase 12 and 15 hydroxyeicosatetraenoic acid (HETE) production. Neither estrogen receptor ERα nor ERβ-specific agonists were able to reproduce the induction of LO expression, but E2-induced expression was effectively blocked by ER non-specific and receptor subtype specific antagonists. Because 12 and 15HETE can increase reactive oxygen species in other cell types, we tested the possibility that E2 could raise ROS through LO. Indeed, E2 as well as the LO products 12 and 15HETE increased reactive oxygen species (ROS) in VSMC. E2-dependent and HETE-induced ROS could be blocked by NAD (P) H-oxidase inhibitors and by the ER general antagonist ICI. E2-induced ROS was partially (∼50%) blocked by the LO inhibitor baicalein, but the LO blocker had no effect on 12 or 15HETE- induced ROS formation, thus suggesting that part of E2-dependent ROS generation resulted from E2-induced 12 and 15HETE. Collectively these findings unveil an unrecognized effect of E2 in human VSMC, to induce 12 and 15LO type 2 expression and activity and suggest that E2-dependent ROS formation in VSMC may be partially mediated by the induction of 12 and 15HETE.

Commentaries on Viewpoint: A call for research to assess and promote functional resilience in astronaut crews

Commentaries on Viewpoint: A call for research to assess and promote functional resilience in astronaut crews

Cryptotanshinone inhibits TNF-α-induced LOX-1 expression by suppressing reactive oxygen species (ROS) formation in endothelial cells.

Cryptotanshinone (CPT) is a natural compound isolated from traditional Chinese medicine Salvia miltiorrhiza Bunge. In the present study, the regulatory effect and potential mechanisms of CPT on tumor necrosis factor alpha (TNF-α) induced lectin-like receptor for oxidized low density lipoprotein (LOX-1) were investigated. Human umbilical vein endothelial cells (HUVECs) were cultured and the effect of TNF-α on LOX-1 expression at mRNA and protein levels was determined by Real-time PCR and Western blotting respectively. The formation of intracellular ROS was determined with fluorescence probe CM-DCFH2-DA. The endothelial ox-LDL uptake was evaluated with DiI-ox-LDL. The effect of CPT on LOX-1 expression was also evaluated with SD rats. TNF-α induced LOX-1 expression in a dose- and time-dependent manner in endothelial cells. TNF-α induced ROS formation, phosphorylation of NF-κB p65 and ERK, and LOX-1 expression, which were suppressed by rotenone, DPI, NAC, and CPT. NF-κB inhibitor BAY11-7082 and ERK inhibitor PD98059 inhibited TNF-α-induced LOX-1 expression. CPT and NAC suppressed TNF-α-induced LOX-1 expression and phosphorylation of NF-κB p65 and ERK in rat aorta. These data suggested that TNF-α induced LOX-1 expression via ROS activated NF-κB/ERK pathway, which could be inhibited by CPT. This study provides new insights for the anti-atherosclerotic effect of CPT.

Exploring the redox balance inside gram-negative bacteria with redox-sensitive GFP

Aerobic bacteria are continuously fighting potential oxidative stress due to endogenous and exogenous reactive oxygen species (ROS). To achieve this goal, bacteria possess a wide array of defenses and stress responses including detoxifying enzymes like catalases and peroxidases; however until now, the dynamics of the intra-bacterial redox balance remained poorly understood. Herein, we used redox-sensitive GFP (roGFP2) inside a variety of gram-negative bacteria to study real-time redox dynamics immediately after a challenge with hydrogen peroxide. Using this biosensor, we determined the individual contributions of catalases and peroxidases and found that each enzyme contributes more to rapid detoxification or to prolonged catalytic activity. We also found that the total catalytic power is affected by environmental conditions. Additionally, using a Salmonella strain that is devoid of detoxifying enzymes, we examined endogenous ROS production. By measuring endogenous ROS production, we assessed the role of oxidative stress in toxicity of heavy metals and antibiotics. We found that exposure to nickel induced significant oxidative stress whereas cobalt (which was previously implicated to induce oxidative stress) did not induce ROS formation. Since a turbulent debate evolves around oxidative stress as a general killing mechanism by antibiotics (aminoglycosides, fluoroquinolones and β-lactams), we measured oxidative stress in bacteria that were challenged with these antibiotics. Our results revealed that antibiotics do not induce ROS formation in bacteria thereby disputing a role for oxidative stress as a general killing mechanism. Together, our results expose how the intra-bacterial redox balance in individual microorganisms is affected by environmental conditions and encounters with stress-inducing compounds. These findings demonstrate the significant potential of roGFP2 as a redox biosensor in gram-negative bacteria to investigate redox dynamics under a variety of circumstances.

Abstract 268: Adiponectin Ameliorates Palmitate Acid Induced Endothelial Inflammation in Endothelial Cells

Endothelial dysfunction (ED) is considered an early event of cardiovascular diseases including hypertension, atherosclerosis and so on. Inflammation participates centrally in all stages of cardiovascular diseases and is considered as a hallmark of endothelial dysfunction. In this study, the effect of adiponectin (APN), an adipocytokine derived mainly from adipocytes, on palmitate acid (PA)-induced inflammation in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were treated with PA with or without APN pretreatment. The mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and ICAM-1 were measured with RT-PCR. The protein expression of ICAM-1, NOX1, NOX2, NOX4, and phosphorylation of MAPKs (JNK, ERK, and p38MAPK), IKKβ, p65 NF-κB were determined by Western blotting. Intracellular reactive oxygen species (ROS) and nitric oxide (NO) formation were determined with DCFH2-DA and DAF-FM respectively. APN significantly ameliorated PA-induced mRNA expression of TNF-α, IL-6 and ICAM-1 and protein expression of ICAM-1, NOX2, and phosphorylation of IKKβ, p65 NF-κB, p38MAPK, without affecting NOX2 and phosphorylation of JNK and ERK. APN also partly reversed PA induced ROS formation and NO decrease. NAC, a ROS scavenger, showed similar activities. The p38MAK inhibitor, SB203580, also reversed PA induced protein expression of ICAM-1 and mRNA expression of TNF-α, IL-6 and ICAM-1. Taken together, these results showed that APN improved PA induced endothelial dysfunction by regulating ROS/p38MAK/NF-κB pathways. Acknowledgement: This study was supported by the National Natural Science Foundation of China (No. 81160048) and the Science and Technology Development Fund of Macau Special Administrative Region (No. 021/2012/A1).

Assessing reproductive toxicity of two environmental toxicants with a novel in vitro human spermatogenic model

Environmental influences and insults by reproductive toxicant exposure can lead to impaired spermatogenesis or infertility. Understanding how toxicants disrupt spermatogenesis is critical for determining how environmental factors contribute to impaired fertility. While current animal models are available, understanding of the reproductive toxic effects on human fertility requires a more robust model system. We recently demonstrated that human pluripotent stem cells can differentiate into spermatogonial stem cells/spermatogonia, primary and secondary spermatocytes, and haploid spermatids; a model that mimics many aspects of human spermatogenesis. Here, using this model system, we examine the effects of 2-bromopropane (2-BP) and 1,2,dibromo-3-chloropropane (DBCP) on in vitro human spermatogenesis. 2-BP and DBCP are non-endocrine disrupting toxicants that are known to impact male fertility. We show that acute treatment with either 2-BP or DBCP induces a reduction in germ cell viability through apoptosis. 2-BP and DBCP affect viability of different cell populations as 2-BP primarily reduces spermatocyte viability, whereas DBCP exerts a much greater effect on spermatogonia. Acute treatment with 2-BP or DBCP also reduces the percentage of haploid spermatids. Both 2-BP and DBCP induce reactive oxygen species (ROS) formation leading to an oxidized cellular environment. Taken together, these results suggest that acute exposure with 2-BP or DBCP causes human germ cell death in vitro by inducing ROS formation. This system represents a unique platform for assessing human reproductive toxicity potential of various environmental toxicants in a rapid, efficient, and unbiased format.

Organic extracts from African dust storms stimulate oxidative stress and induce inflammatory responses in human lung cells through Nrf2 but not NF-κB

The health impact of the global African dust event (ADE) phenomenon in the Caribbean has been vaguely investigated. Heavy metals in ADE and non-ADE extracts were evaluated for the formation of reactive oxygen species (ROS) and antioxidant capacity by cells using, deferoxamine mesylate (DF) and N-acetyl-l-cysteine (NAC). Results show that ADE particulate matter 2.5 (PM2.5) induces ROS and stimulates oxidative stress. Pre-treatment with DF reduces ROS in ADE and Non-ADE extracts and in lung cells demonstrating that heavy metals are of utmost importance. Glutathione-S-transferase and Heme Oxygenase 1 mRNA levels are induced with ADE PM and reduced by DF and NAC. ADE extracts induced Nrf2 activity and IL-8 mRNA levels significantly more than Non-ADE. NF-κB activity was not detected in any sample. Trace elements and organic constituents in ADE PM2.5 enrich the local environment load, inducing ROS formation and activating antioxidant-signaling pathways increasing pro-inflammatory mediator expressions in lung cells.

A small library of synthetic di-substituted 1, 4-naphthoquinones induces ROS-mediated cell death in murine fibroblasts.

Synthesis of compound libraries and their concurrent assessment as selective reagents for probing and modulating biological function continues to be an active area of chemical biology. Microwave-assisted solid-phase Dötz benzannulation reactions have been used to inexpensively synthesize 2, 3-disubstituted-1, 4-naphthoquinone derivatives. Herein, we report the biological testing of a small library of such compounds using a murine fibroblast cell line (L929). Assessment of cellular viability identified three categories of cytotoxic compounds: no toxicity, low/intermediate toxicity and high toxicity. Increased levels of Annexin-V-positive staining and of caspase 3 activity confirmed that low, intermediate, and highly toxic compounds promote cell death. The compounds varied in their ability to induce mitochondrial depolarization and formation of reactive oxygen species (ROS). Both cytotoxic and non-cytotoxic compounds triggered mitochondrial depolarization, while one highly cytotoxic compound did not. In addition, all cytotoxic compounds promoted increased intracellular ROS but the cells were only partially protected from compound-induced apoptosis when in the presence of superoxide dismutase, catalase, or ascorbic acid suggesting utilization of additional pro-death mechanisms. In summary, nine of twelve (75%) 1, 4-naphthoquinone synthetic compounds were cytotoxic. Although the mitochondria did not appear to be a central target for induction of cell death, all of the cytotoxic compounds induced ROS formation. Thus, the data demonstrate that the synthesis regime effectively created cytotoxic compounds highlighting the potential use of the regime and its products for the identification of biologically relevant reagents.

Extracorporeal shockwaves (ESWs) enhance the osteogenic medium-induced differentiation of adipose-derived stem cells into osteoblast-like cells.

Human adipose-derived stem cells (hASCs) are a promising cell type for bone tissue engineering, given their potential to differentiate into osteoblast-like cells. Interactions among biochemical and mechanical signals result in bone formation and repair. In this process stem cells have a crucial role. Extracorporeal shockwaves (ESWs) are acoustic waves capable of enhancing bone regeneration, suggesting that ESWs may induce some signals for mesenchymal progenitor maturation. The aim of the present work is to investigate the effects of ESW treatment on the differentiation of hASCs into osteoblast-like cells and to better clarify the mechanisms involved. The hASCs were treated with ESWs and osteogenic medium, and the effects in terms of gene expression, alkaline phosphatase (ALP) activity and calcium deposition were then evaluated. Moreover, to investigate the mechanisms of ESW action, reactive oxygen species (ROS) production, extracellular-signal-regulated kinase (ERK) and small 'mothers against' decapentaplegic (Smad) phosphorylation, and bone morphogenetic protein 2 (BMP2) expression were assessed. The ESW treatment increased Runt-related transcription factor 2 (Runx2), ALP and BMP2 expression, as well as ALP activity and calcium deposits with respect to untreated cells. Moreover ESWs induced ROS formation, and both ERK and Smad phosphorylation. The present study shows the effects of ESWs on osteogenic differentiation in an in vitro model using hASCs and defines the mechanisms involved in this process. The observations suggest that the combination of autologous hASCs and ESW treatment may improve bone tissue repair in tissue engineering procedures. Copyright © 2014 John Wiley & Sons, Ltd.

Improved bioactivity of antimicrobial peptides by addition of amino-terminal copper and nickel (ATCUN) binding motifs.

Antimicrobial peptides (AMPs) are promising candidates to help circumvent antibiotic resistance, which is an increasing clinical problem. Amino-terminal copper and nickel (ATCUN) binding motifs are known to actively form reactive oxygen species (ROS) upon metal binding. The combination of these two peptidic constructs could lead to a novel class of dual-acting antimicrobial agents. To test this hypothesis, a set of ATCUN binding motifs were screened for their ability to induce ROS formation, and the most potent were then used to modify AMPs with different modes of action. ATCUN binding motif-containing derivatives of anoplin (GLLKRIKTLL-NH2), pro-apoptotic peptide (PAP; KLAKLAKKLAKLAK-NH2), and sh-buforin (RAGLQFPVGRVHRLLRK-NH2) were synthesized and found to be more active than the parent AMPs against a panel of clinically relevant bacteria. The lower minimum inhibitory concentration (MIC) values for the ATCUN-anoplin peptides are attributed to the higher pore-forming activity along with their ability to cause ROS-induced membrane damage. The addition of the ATCUN motifs to PAP also increases its ability to disrupt membranes. DNA damage is the major contributor to the activity of the ATCUN-sh-buforin peptides. Our findings indicate that the addition of ATCUN motifs to AMPs is a simple strategy that leads to AMPs with higher antibacterial activity and possibly to more potent, usable antibacterial agents.

Isolation, identification and characterization of highly tellurite-resistant, tellurite-reducing bacteria from Antarctica

The tellurium oxyanion, tellurite, is extremely noxious to most living organisms. Its toxicity has been mainly related to the generation of reactive oxygen species (ROS) as well as to an unbalancing of the thiol:redox buffering system. Nevertheless, a few bacteria are capable of thriving at high tellurite concentrations. One mechanism of resistance is the enzymatic and non-enzymatic reduction of tellurite to the less toxic elemental tellurium. This reduction generates nano- to micrometric tellurium crystals that display different shapes and sizes.To date, a very limited number of highly tellurite-resistant and tellurite-reducing bacterial species are available from international culture collections. In this work, we decided to look for tellurite-reducing bacteria from an extreme environment, Antarctica. This environment exhibits a combination of several extreme factors such as high UV-radiation and desiccation and freezing conditions that impact directly on the local biodiversity. Since, as does, all these factors induce ROS formation, we hypothesized that Antarctic bacteria could also exhibit tellurite-resistance. In this context, we isolated 123 tellurite-resistant bacteria, and characterized six new tellurite-resistant and tellurite-reducing bacterial strains from samples collected in Antarctica. These strains were identified according to their 16S rRNA gene sequence as Staphylococcus hameolyticus, Staphylococcus sciuri, Acinetobacter haemolyticus, Pseudomonas lini, and two strains of Psychrobacter immobilis.The isolates display tellurite-resistance about 35- to 500-fold higher than Escherichia coli (Te-sensitive organism), and a high level of tellurite reduction which might be interesting for an application in the field of bioremediation or nanoparticle biosynthesis.

Ridaifen-SB8, a novel tamoxifen derivative, induces apoptosis via reactive oxygen species-dependent signaling pathway

Tamoxifen is an anticancer agent widely used for treatment of estrogen receptor (ERα)-positive breast cancer. We previously developed a novel synthesis of tamoxifen and its derivatives, named Ridaifens (RIDs). Some of them, including RID-SB8, exhibited a stronger anticancer activity than tamoxifen in ERα-positive MCF-7 cells while having lost the affinity for ERα, suggesting an ERα-independent anticancer mode of action. In this study, we investigated the underlying mechanism by which RID-SB8 exerts anticancer activity. As expected, anticancer activity of RID-SB8 was not influenced upon knockdown of ERα expression in MCF-7 cells. RID-SB8 exerted similar anticancer effects on thirteen ERα-negative cancer cell lines including human gliosarcoma SF539 cells. In SF539 cells, RID-SB8 triggered loss of mitochondrial membrane potential (ΔΨm) and progression of apoptosis accompanied by activation of caspases and translocation of apoptosis-inducing factor (AIF) to the nucleus. Furthermore, it induced reactive oxygen species (ROS), and a ROS scavenger, N-acetylcysteine (NAC), canceled loss of ΔΨm and progression of apoptosis triggered by RID-SB8. Using fifteen human cancer cell lines, we demonstrated a significant correlation between RID-SB8 concentration required for ROS production and that required for cytotoxic effect across these cell lines, but such correlation was not observed for tamoxifen. Finally, the selective induction of ROS and cytotoxic effect on cancer cells by RID-SB8 were confirmed. From these results, we concluded that RID-SB8 exerts an anticancer effect via a mode of action distinct from tamoxifen, and that RID-SB8 could become a promising anticancer lead compound which selectively induces ROS formation and apoptosis in cancer cells.

Cyclosporine A Cardioprotection

Cyclosporine A Cardioprotection

Ablation of Ceramide Synthase 2 Causes Chronic Oxidative Stress Due to Disruption of the Mitochondrial Respiratory Chain

Ceramide is a key intermediate in the pathway of sphingolipid biosynthesis and is an important intracellular messenger. We recently generated a ceramide synthase 2 (CerS2) null mouse that cannot synthesize very long acyl chain (C22-C24) ceramides. This mouse displays severe and progressive hepatopathy. Significant changes were observed in the sphingolipid profile of CerS2 null mouse liver, including elevated C16-ceramide and sphinganine levels in liver and in isolated mitochondrial fractions. Because ceramide may be involved in reactive oxygen species (ROS) formation, we examined whether ROS generation was affected in CerS2 null mice. Levels of a number of anti-oxidant enzymes were elevated, as were lipid peroxidation, protein nitrosylation, and ROS. ROS were generated from mitochondria due to impaired complex IV activity. C16-ceramide, sphingosine, and sphinganine directly inhibited complex IV activity in isolated mitochondria and in mitoplasts, whereas other ceramide species, sphingomyelin, and diacylglycerol were without effect. A fluorescent analog of sphinganine accumulated in mitochondria. Heart mitochondria did not display a substantial alteration in the sphingolipid profile or in complex IV activity. We suggest that C16-ceramide and/or sphinganine induce ROS formation through the modulation of mitochondrial complex IV activity, resulting in chronic oxidative stress. These results are of relevance for understanding modulation of ROS signaling by sphingolipids.

P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia

The P2X7 purinergic receptor is a ligand-gated cation channel expressed on leukocytes including microglia. This study aimed to determine if P2X7 activation induces the uptake of organic cations, reactive oxygen species (ROS) formation, and death in the murine microglial EOC13 cell line. Using the murine macrophage J774 cell line as a positive control, RT-PCR, immunoblotting, and immunolabelling established the presence of P2X7 in EOC13 cells. A cytofluorometric assay demonstrated that the P2X7 agonists adenosine-5′-triphosphate (ATP) and 2′(3′)-O-(4-benzoylbenzoyl) ATP induced ethidium+ or YO-PRO-12+ uptake into both cell lines. ATP induced ethidium+ uptake into EOC13 cells in a concentration-dependent manner, with an EC50 of ~130 μM. The P2X7 antagonists Brilliant Blue G, A438079, AZ10606120, and AZ11645373 inhibited ATP-induced cation uptake into EOC13 cells by 75–100%. A cytofluorometric assay demonstrated that P2X7 activation induced ROS formation in EOC13 cells, via a mechanism independent of Ca2+ influx and K+ efflux. Cytofluorometric measurements of Annexin-V binding and 7AAD uptake demonstrated that P2X7 activation induced EOC13 cell death. The ROS scavenger N-acetyl-L-cysteine impaired both P2X7-induced EOC13 ROS formation and cell death, suggesting that ROS mediate P2X7-induced EOC13 death. In conclusion, P2X7 activation induces the uptake of organic cations, ROS formation, and death in EOC13 microglia.

Platelet-derived growth factor triggers PKA-mediated signalling by a redox-dependent mechanism in rat renal mesangial cells

Inflammatory glomerular kidney diseases are often accompanied with a massive production of reactive oxygen species (ROS) that affect the function of the glomerular filtration barrier and contribute to mesangiolysis via the induction of cell death in mesangial cells. Intriguingly, ROS also trigger fine-tuned signalling processes that affect gene expression and cell proliferation or migration. To define such redox-driven signalling devices, a proteomics approach was performed to identify the formation of protein complexes induced by ROS. To this end, protein lysates of human podocytes were treated with or without hydrogen peroxide (250μM). Thereafter cell lysates were subjected to diagonal 2D gel electrophoresis and putative redox-affected proteins were analysed by MS/MS analysis. Among others, the regulatory subunit of protein kinase A (PKA) could be identified that forms homodimers under oxidative conditions. To evaluate whether ROS dependent dimerization of PKA also occurs in a more physiological setting, rat mesangial cells were treated with platelet-derived growth factor-BB (PDGF-BB) to induce ROS formation. This regimen resulted in a redox dependent dimerization of the R-subunits of PKA. To demonstrate whether PDGF-BB induced ROS formation affects PKA dependent pathways, the effects of PDGF-BB on phosphorylation of serine 157 of vasodilator stimulated protein (VASP) a classical target of PKA were analysed. Interestingly PDGF-BB induced VASP phosphorylation in a ROS dependent manner but independent of changes in cAMP levels. Taken together, we demonstrate a redox-mediated activation of PKA by PDGF-BB thus highlighting a physiological role of ROS as regulator of PKA activity in rat mesangial cells.

P2X7 receptor activation induces reactive oxygen species formation in erythroid cells

The presence of P2X7 on erythroid cells is well established, but its physiological role remains unclear. The current study aimed to determine if P2X7 activation induces reactive oxygen species (ROS) formation in murine erythroleukaemia (MEL) cells, a commonly used erythroid cell line. ATP induced ROS formation in a time- and concentration-dependent fashion. The most potent P2X7 agonist, 2'(3')-O-(4-benzoylbenzoyl)ATP, but not UTP or ADP, also induced ROS formation. The P2X7 antagonist, A-438079, impaired ATP-induced ROS formation. The ROS scavenger, N-acetyl-L-cysteine, and the ROS inhibitor, diphenyleneiodonium, also impaired P2X7-induced ROS formation, but use of enzyme-specific ROS inhibitors failed to identify the intracellular source of P2X7-induced ROS formation. P2X7-induced ROS formation was impaired partly by physiological concentrations of Ca(2+) and Mg(2+) and almost completely in cells in N-methyl-D-glucamine chloride medium. The p38 MAPK inhibitors SB202190 and SB203580, and the caspase inhibitor Z-VAD-FMK, but not N-acetyl-L-cysteine, impaired P2X7-induced MEL cell apoptosis. ATP also stimulated p38 MAPK and caspase activation, both of which could be impaired by A-438079. In conclusion, these findings indicate that P2X7 activation induces ROS formation in MEL cells and that this process may be involved in events downstream of P2X7 activation, other than apoptosis, in erythroid cells.

A new water-soluble Cu(II) chelator that retrieves Cu from Cu(amyloid-β) species, stops associated ROS production and prevents Cu(II)‐induced Aβ aggregation

The synthesis of the H2L2− ligand (N,N′-Bis[(5-sulfonato-2-hydroxy)benzyl]-N,N′-dimethyl-ethane-1,2-diamine) and characterizations of the corresponding Cu(II) complex [Cu(L)(H2O)]2− (1) by X-ray diffraction, EPR, UV–Visible and potentiometry are described. At pH 7.4, the affinity of Cu(II) for this ligand is approximately 4×1014M−1. Coordination of redox active metal ions such as copper or iron to the amyloid-β (Aβ) peptide has been linked to deleterious processes encountered in the etiology of Alzheimer disease (AD), such as Aβ aggregation and reactive oxygen species (ROS) production. In this context, the ability of the H2L2− to extract Cu(II) from Cu(Aβ) species where Aβ is the peptide involved in AD, is reported as well as its capacity to redox silence the Cu(Aβ) induced ROS formation and to prevent Cu(II)‐induced Aβ aggregation. Such water soluble sulfonato-derivatives of Cu(II) chelators are very interesting counterparts for in vitro study of chelators' properties required to attend further biological applications as therapeutic tools against AD.

Oxidatively Truncated Phospholipids Are Required Agents of Tumor Necrosis Factor α (TNFα)-induced Apoptosis

TNFα generates reactive oxygen species (ROS) at the cell surface that induce cell death, but how ROS communicate to mitochondria and their specific apoptotic action(s) are both undefined. ROS oxidize phospholipids to hydroperoxides that are friable and fragment adjacent to the (hydro)peroxide function, forming truncated phospholipids, such as azelaoyl phosphatidylcholine (Az-PC). Az-PC is relatively soluble, and exogenous Az-PC rapidly enters cells to damage mitochondrial integrity and initiate intrinsic apoptosis. We determined whether this toxic phospholipid is formed within cells during TNFα stimulation in sufficient quantities to induce apoptosis and if they are essential in TNFα-induced cytotoxicity. We found that TNFα induced ROS formation and phospholipid peroxidation in Jurkat cells, and either chemical interference with NADPH oxidase activity or siRNA suppression of the NADPH oxidase-4 subunit blocked ROS accumulation and phospholipid peroxidation. Mass spectrometry showed that phospholipid peroxides and then Az-PC increased after TNFα exposure, whereas ROS inhibition abolished Az-PC accumulation and TNFα-induced cell death. Glutathione peroxidase-4 (GPx4), which specifically metabolizes lipid hydroperoxides, fell in TNFα-stimulated cells prior to death. Ectopic GPx4 overcame this, reduced peroxidized phospholipid accumulation, blocked Az-PC accumulation, and prevented death. Conversely, GPx4 siRNA knockdown enhanced phospholipid peroxidation, increasing TNFα-stimulated Az-PC formation and apoptosis. Truncated phospholipids were essential elements of TNFα-induced apoptosis because overexpression of PAFAH2 (a phospholipase A(2) that selectively hydrolyzes truncated phospholipids) blocked TNFα-induced Az-PC accumulation without affecting phospholipid peroxidation. PAFAH2 also abolished apoptosis. Thus, phospholipid oxidation and truncation to apoptotic phospholipids comprise an essential element connecting TNFα receptor signaling to mitochondrial damage and apoptotic death.

Involvement of four different intracellular sites in chloroacetaldehyde- induced oxidative stress cytotoxicity.

Chloroacetaldehyde (CAA) is a chlorination by-product in finished drinking water and a toxic metabolite of a wide variety of industrial chemicals (e.g. vinyl chloride) and chemotherapeutic agents (e.g. cyclophosphamide and ifosfamide). In this research, the cytotoxic mechanisms of CAA in freshly isolated rat hepatocytes were investigated.CAA cytotoxicity was associated with reactive oxygen species (ROS) formation and glutathione depletion suggesting that oxidative stress contributed to the CAA cytotoxic mechanism. CAA-induced oxidative stress cytotoxicity markers were significantly prevented by antioxidants, ROS scavengers, mitochondrial permeability transition (MPT) pore sealing agents, endocytosis inhibitors, ATP generators and xanthine oxidase inhibitor. In our study the hepatocyte mitochondrial membrane potential was rapidly decreased by CAA which was prevented by antioxidants and ROS scavenger indicating that mitochondrial membrane damage was a consequence of ROS formation. CAA cytotoxicity was also associated with lysosomal membrane rupture. OUR FINDINGS SHOWED THAT AT LEAST FOUR DIFFERENT INTRACELLULAR SOURCES INCLUDING: metabolic enzymes cytochrome P450 and xanthine oxidase, mitochondrial respiratory chain disruption and lysosomal Haber-weiss reaction, were involved in CAA induced ROS formation and other subsequent cytotoxic events. Our other interesting finding was that the lysosomotropic agents prevented CAA induced mitochondrial membrane potential collapse and mitochondrial MPT pore sealing agents inhibited lysosomal membrane damage caused by CAA. It can therefore be suggested that there is probably a toxic interaction (cross-talk) between mitochondrial and lysosomal oxidative stress generating systems, which potentiates each organelle damage and ROS formation in CAA- induced hepatotoxicity.