Hypothyroidism-associated missense mutation impairs NADPH oxidase activity and intracellular trafficking of Duox2

In the thyroid gland Duox2-derived H₂O₂ is essential for thyroid hormone biosynthesis. Several patients were identified with partial or severe iodide organification defects caused by mutation in the gene for Duox2 or its maturation factor, DuoxA2. A Duox2-deficient (Duox2^thyd) mouse model enabled in vivo investigation of its critical function in thyroid tissues, but its roles proposed in host defense or other innate responses in nonthyroid tissues remain less certain. These mice carry a spontaneous DUOX2 missense mutation, a T→G transversion, in exon 16 that changes the highly conserved valine 674 to glycine and results in severe congenital hypothyroidism. The exact mechanism underlying the effects of the V674G mutation has not been elucidated at the molecular or cellular level. To determine how the V674G mutation leads to congenital hypothyroidism, we introduced the same mutation into human Duox2 or Duox1 cDNAs and expressed them in HEK-293 cells stably expressing the corresponding DuoxA proteins. We found that the valine→glycine mutant Duox proteins fail to produce H₂O₂, lose their plasma membrane localization pattern, and are retained within the endoplasmic reticulum. The Duox2 mutant binds to DuoxA2, but appears to be unstable owing to this retention. Immunohistochemical staining of Duox2 in murine salivary gland ducts showed that Duox2 in mutant mice loses its condensed apical plasma membrane localization pattern characteristic of wild-type Duox2 and accumulates in punctate vesicular structures within cells. Our findings demonstrate that changing the highly conserved valine 674 in Duox2 leads to impaired subcellular targeting and reactive oxygen species release required for hormonogenesis, resulting in congenital hypothyroidism.     

Characterization of hydrogen peroxide production by Duox in bronchial epithelial cells exposed to Pseudomonas aeruginosa

Hydrogen peroxide production by the NADPH oxidase Duox1 occurs during activation of respiratory epithelial cells stimulated by purified bacterial ligands, such as lipopolysaccharide. Here, we characterize Duox activation using intact bacterial cells of several airway pathogens. We found that only Pseudomonas aeruginosa, not Burkholderia cepacia or Staphylococcus aureus, triggers H₂O₂ production in bronchial epithelial cells in a calcium-dependent but predominantly ATP-independent manner. Moreover, by comparing mutant Pseudomonas strains, we identify several virulence factors that participate in Duox activation, including the type-three secretion system. These data provide insight on Duox activation by mechanisms unique to P. aeruginosa.     

Oxidative epithelial host defense is regulated by infectious and inflammatory stimuli

Epithelia express oxidative antimicrobial protection that uses lactoperoxidase (LPO), hydrogen peroxide (H₂O₂), and thiocyanate to generate the reactive hypothiocyanite. Duox1 and Duox2, found in epithelia, are hypothesized to provide H₂O₂ for use by LPO. To investigate the regulation of oxidative LPO-mediated host defense by bacterial and inflammatory stimuli, LPO and Duox mRNA were followed in differentiated primary human airway epithelial cells challenged with Pseudomonas aeruginosa flagellin or IFN-γ. Flagellin upregulated Duox2 mRNA 20-fold, but upregulated LPO mRNA only 2.5-fold. IFN-γ increased Duox2 mRNA 127-fold and upregulated LPO mRNA 10-fold. DuoxA2, needed for Duox2 activity, was also upregulated by flagellin and IFN-γ. Both stimuli increased H₂O₂ synthesis and LPO-dependent killing of P. aeruginosa. Reduction of Duox1 by siRNA showed little effect on basal H₂O₂ production, whereas Duox2 siRNA markedly reduced basal H₂O₂ production and resulted in an 8-fold increase in Nox4 mRNA. In conclusion, large increases in Duox2-mediated H₂O₂ production seem to be coordinated with increases in LPO mRNA and, without increased LPO, H₂O₂ levels in airway secretion are expected to increase substantially. The data suggest that Duox2 is the major contributor to basal H₂O₂ synthesis despite the presence of greater amounts of Duox1.     

Nox4 and Duox1/2 Mediate Redox Activation of Mesenchymal Cell Migration by PDGF

Platelet derived growth factor (PDGF) orchestrates wound healing and tissue regeneration by regulating recruitment of the precursor mesenchymal stromal cells (MSC) and fibroblasts. PDGF stimulates generation of hydrogen peroxide that is required for cell migration, but the sources and intracellular targets of H₂O₂ remain obscure. Here we demonstrate sustained live responses of H₂O₂ to PDGF and identify PKB/Akt, but not Erk1/2, as the target for redox regulation in cultured 3T3 fibroblasts and MSC. Apocynin, cell-permeable catalase and LY294002 inhibited PDGF-induced migration and mitotic activity of these cells indicating involvement of PI3-kinase pathway and H₂O₂. Real-time PCR revealed Nox4 and Duox1/2 as the potential sources of H₂O₂. Silencing of Duox1/2 in fibroblasts or Nox4 in MSC reduced PDGF-stimulated intracellular H₂O₂, PKB/Akt phosphorylation and migration, but had no such effect on Erk1/2. In contrast to PDGF, EGF failed to increase cytoplasmic H₂O₂, phosphorylation of PKB/Akt and migration of fibroblasts and MSC, confirming the critical impact of redox signaling. We conclude that PDGF-induced migration of mesenchymal cells requires Nox4 and Duox1/2 enzymes, which mediate redox-sensitive activation of PI3-kinase pathway and PKB/Akt.     

Genetic Mechanisms Underlying the Pathogenicity of Cold-Stressed Salmonella enterica Serovar Typhimurium in Cultured Intestinal Epithelial Cells

Salmonella encounters various stresses in the environment and in the host during infection. The effects of cold (5°C, 48 h), peroxide (5 mM H₂O₂, 5 h) and acid stress (pH 4.0, 90 min) were tested on pathogenicity of Salmonella. Prior exposure of Salmonella to cold stress significantly (P < 0.05) increased adhesion and invasion of cultured intestinal epithelial (Caco-2) cells. This increased Salmonella-host cell association was also correlated with significant induction of several virulence-associated genes, implying an increased potential of cold-stressed Salmonella to cause an infection. In Caco-2 cells infected with cold-stressed Salmonella, genes involved in the electron transfer chain were significantly induced, but no simultaneous significant increase in expression of antioxidant genes that neutralize the effect of superoxide radicals or reactive oxygen species was observed. Increased production of caspase 9 and caspase 3/7 was confirmed during host cell infection with cold-stressed Salmonella. Further, a prophage gene, STM2699, induced in cold-stressed Salmonella and a spectrin gene, SPTAN1, induced in Salmonella-infected intestinal epithelial cells were found to have a significant contribution in increased adhesion and invasion of cold-stressed Salmonella in epithelial cells.     

Urothelial cells produce hydrogen peroxide through the activation of Duox1

Hydrogen peroxide (H₂O₂) has important messenger and effector functions in the plant and animal kingdom. Phagocytes produce H₂O₂ to kill pathogens, and epithelial cells of large airways have also been reported to produce H₂O₂ for signaling and host defense purposes. In this report, we show for the first time that urothelial cells produce H₂O₂ in response to a calcium signal. Using a gene-deficient mouse model we also demonstrate that H₂O₂ is produced by the NADPH oxidase Duox1, which is expressed in the mouse urothelium. In contrast, we found no evidence for the expression of lactoperoxidase, an enzyme that has been shown to cooperate with Duox enzymes. We also found that specific activation of TRPV4 calcium channels elicits a calcium signal and stimulates H₂O₂ production in urothelial cells. Furthermore, we detected altered pressure responses in the urinary bladders of Duox1 knockout animals. Our results raise the possibility that mechanosensing in epithelial cells involves calcium-dependent H₂O₂ production similar to that observed in plants.     

The Extracellular A-loop of Dual Oxidases Affects the Specificity of Reactive Oxygen Species Release

NADPH oxidase (Nox) family proteins produce superoxide (O₂^⨪) directly by transferring an electron to molecular oxygen. Dual oxidases (Duoxes) also produce an O₂^⨪ intermediate, although the final species secreted by mature Duoxes is H₂O₂, suggesting that intramolecular O₂^⨪ dismutation or other mechanisms contribute to H₂O₂ release. We explored the structural determinants affecting reactive oxygen species formation by Duox enzymes. Duox2 showed O₂^⨪ leakage when mismatched with Duox activator 1 (DuoxA1). Duox2 released O₂^⨪ even in correctly matched combinations, including Duox2 + DuoxA2 and Duox2 + N-terminally tagged DuoxA2 regardless of the type or number of tags. Conversely, Duox1 did not release O₂^⨪ in any combination. Chimeric Duox2 possessing the A-loop of Duox1 showed no O₂^⨪ leakage; chimeric Duox1 possessing the A-loop of Duox2 released O₂^⨪. Moreover, Duox2 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA2 showed enhanced O₂^⨪ release, and Duox1 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA1 acquired O₂^⨪ leakage. Although we identified Duox1 A-loop residues (His¹⁰⁷¹, His¹⁰⁷², and Gly¹⁰⁷⁴) important for reducing O₂^⨪ release, mutations of these residues to those of Duox2 failed to convert Duox1 to an O₂^⨪-releasing enzyme. Using immunoprecipitation and endoglycosidase H sensitivity assays, we found that the A-loop of Duoxes binds to DuoxA N termini, creating more stable, mature Duox-DuoxA complexes. In conclusion, the A-loops of both Duoxes support H₂O₂ production through interaction with corresponding activators, but complex formation between the Duox1 A-loop and DuoxA1 results in tighter control of H₂O₂ release by the enzyme complex.     

Mass spectrometry-based quantitative proteomic analysis of <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Enteritidis protein expression upon exposure to hydrogen peroxide

Background  

Salmonella enterica, a common food-borne bacterial pathogen, is believed to change its protein expression profile in the presence of different environmental stress such as that caused by the exposure to hydrogen peroxide (H₂O₂), which can be generated by phagocytes during infection and represents an important antibacterial mechanism of host cells. Among Salmonella proteins, the effectors of Salmonella pathogenicity island 1 and 2 (SPI-1 and SPI-2) are of particular interest since they are expressed during host infection in vivo and are important for invasion of epithelial cells and for replication in organs during systemic infection, respectively. However, the expression profiles of these proteins upon exposure to H₂O₂ or to host cells in vivo during the established phase of systemic infection have not been extensively studied.     

Ovarian Dual Oxidase (Duox) Activity Is Essential for Insect Eggshell Hardening and Waterproofing

In insects, eggshell hardening involves cross-linking of chorion proteins via their tyrosine residues. This process is catalyzed by peroxidases at the expense of H₂O₂ and confers physical and biological protection to the developing embryo. Here, working with Rhodnius prolixus, the insect vector of Chagas disease, we show that an ovary dual oxidase (Duox), a NADPH oxidase, is the source of the H₂O₂ that supports dityrosine-mediated protein cross-linking and eggshell hardening. RNAi silencing of Duox activity decreased H₂O₂ generation followed by a failure in embryo development caused by a reduced resistance to water loss, which, in turn, caused embryos to dry out following oviposition. Phenotypes of Duox-silenced eggs were reversed by incubation in a water-saturated atmosphere, simultaneous silencing of the Duox and catalase genes, or H₂O₂ injection into the female hemocoel. Taken together, our results show that Duox-generated H₂O₂ fuels egg chorion hardening and that this process plays an essential role during eggshell waterproofing.     

Micromolar concentrations of hydrogen peroxide induce oxidative DNA lesions more efficiently than millimolar concentrations in mammalian cells

Reactive oxygen species produce oxidized bases, deoxyribose lesions and DNA strand breaks in mammalian cells. Previously, we demonstrated that aldehydic DNA lesions (ADLs) were induced in mammalian cells by 10 mM hydrogen peroxide (H₂O₂). Interestingly, a bimodal H₂O₂ dose–response relationship in cell toxicity has been reported for Escherichia coli deficient in DNA repair as well as Chinese hamster ovary (CHO) cells. Furthermore, it has been demonstrated that H₂O₂ causes single-strand breaks in purified DNA in the presence of iron and induces mitochondrial DNA damage in CHO cells with a biphasic dose–response curve. Here we show that H₂O₂ produces ADLs at concentrations as low as 0.06 mM in HeLa cells and that lower concentrations of H₂O₂ were much more efficient at inducing ADLs than higher concentrations. This dose–response curve is strikingly similar to that for cell killing effects in E.coli deficient in DNA repair exposed to H₂O₂. Interestingly, serial treatment of submillimolar levels of H₂O₂ induced a massive accumulation of ADLs. The toxicity arising from H₂O₂ determined by intracellular NAD(P)H in cells correlated well with the formation of ADLs. The addition of dipyridyl, an iron (II)-specific chelator, significantly protected against DNA damage and cell toxicity from submillimolar, but not millimolar, amounts of H₂O₂. These results suggest that ADLs induced by submillimolar levels of H₂O₂ may be due to a Fenton-type reaction between H₂O₂ and intracellular iron ions in mammalian cells.     

Salmonella Infection Upregulates the Leaky Protein Claudin-2 in Intestinal Epithelial Cells

Background

Tight junctions seal the space between adjacent epithelial cells. Mounting evidence suggests that tight junction proteins play a key role in the pathogenesis of human disease. Claudin is a member of the tight junction protein family, which has 24 members in humans. To regulate cellular function, claudins interact structurally and functionally with membrane and scaffolding proteins via their cytoplasmic domain. In particular, claudin-2 is known to be a leaky protein that contributes to inflammatory bowel disease and colon cancer. However, the involvement of claudin-2 in bacterial infection in the intestine remains unknown.

Methods/Principal Findings

We hypothesized that Salmonella elevates the leaky protein claudin-2 for its own benefit to facilitate bacterial invasion in the colon. Using a Salmonella-colitis mouse model and cultured colonic epithelial cells, we found that pathogenic Salmonella colonization significantly increases the levels of claudin-2 protein and mRNA in the intestine, but not that of claudin-3 or claudin-7 in the colon, in a time-dependent manner. Immunostaining studies showed that the claudin-2 expression along the crypt-villous axis postinfection. In vitro, Salmonella stimulated claudin-2 expression in the human intestinal epithelial cell lines SKCO15 and HT29C19A. Further analysis by siRNA knockdown revealed that claudin-2 is associated with the Salmonella-induced elevation of cell permeability. Epithelial cells with claudin-2 knockdown had significantly less internalized Salmonella than control cells with normal claudin-2 expression. Inhibitor assays demonstrated that this regulation is mediated through activation of the EGFR pathway and the downstream protein JNK.

Conclusion/Significance

We have shown that Salmonella targets the tight junction protein claudin-2 to facilitate bacterial invasion. We speculate that this disruption of barrier function contributes to a new mechanism by which bacteria interact with their host cells and suggests the possibility of blocking claudin-2 as a potential therapeutic strategy to prevent bacterial invasion.     

Thiol Peroxidase Protects Salmonella enterica from Hydrogen Peroxide Stress In Vitro and Facilitates Intracellular Growth

At present, Salmonella is considered to express two peroxiredoxin-type peroxidases, TsaA and AhpC. Here we describe an additional peroxiredoxin, Tpx, in Salmonella enterica and show that a single tpx mutant is susceptible to exogenous hydrogen peroxide (H₂O₂), that it has a reduced capacity to degrade H₂O₂ compared to the ahpCF and tsaA mutants, and that its growth is affected in activated macrophages. These results suggest that Tpx contributes significantly to the sophisticated defense system that the pathogen has evolved to survive oxidative stress.Salmonella is an important human pathogen which causes a variety of diseases, including gastroenteritis, septicemia, and typhoid fever. In the host, salmonellae reside inside phagocytic cells and are exposed to various host defense mechanisms, including oxidative stress (13). The production of superoxide anion (O₂⁻) is crucial, as individuals with chronic granulomatous disease, which is due to a defective phagocyte NADPH oxidase, are more susceptible to infections with Salmonella (10). Likewise, diminished NADPH oxidase activity leads to increased susceptibility to Salmonella in murine macrophages (20-22, 25). Superoxide anion (O₂⁻) is weakly reactive and fails to pass through the bacterial cell wall. After conversion to H₂O₂ by either spontaneous or enzymatic dismutation by superoxide dismutases, it readily diffuses into the bacterial cell and forms reactive hydroxyl radicals (OH) that damage macromolecules such as DNA, proteins, and lipids (12, 17).In principle, Salmonella possesses two classes of enzymes to degrade H₂O₂. Catalases degrade H₂O₂ to water and molecular oxygen independent of an additional reductant. Peroxiredoxin-type peroxidases (peroxiredoxins) reduce organic hydroperoxides to alcohols and hydrogen peroxide to water at the expense of NADH or NADPH. In a recent study by Hébrard et al., three members of the catalase family, KatG, KatE, and KatN, and two members of the peroxiredoxin family, AhpC and TsaA, were characterized in Salmonella (14). Previously it had been shown that single katE, katG, and katN Salmonella mutants did not show increased susceptibility to exogenous H₂O₂ (3, 24). In macrophages a katG katE katN triple mutant had no growth defect, whereas an ahpCF tsaA double mutant showed a reduced growth rate in macrophages (14). These observations point out the multiple routes that have evolved in Salmonella to protect the pathogen against oxidative stress and suggest that peroxiredoxins play a dominant role in the antioxidant defense during infection. In this study we characterized a third peroxiredoxin-type peroxidase, Tpx. Surprisingly, a simple tpx mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium) was more susceptible to exogenous H₂O₂ than the wild type (WT). The mutant grew less well in activated macrophages and showed a reduced peroxidase activity toward H₂O₂.     

Hydrogen peroxide primes heart regeneration with a derepression mechanism

While the adult human heart has very limited regenerative potential, the adult zebrafish heart can fully regenerate after 20% ventricular resection. Although previous reports suggest that developmental signaling pathways such as FGF and PDGF are reused in adult heart regeneration, the underlying intracellular mechanisms remain largely unknown. Here we show that H₂O₂ acts as a novel epicardial and myocardial signal to prime the heart for regeneration in adult zebrafish. Live imaging of intact hearts revealed highly localized H₂O₂ (∼30 μM) production in the epicardium and adjacent compact myocardium at the resection site. Decreasing H₂O₂ formation with the Duox inhibitors diphenyleneiodonium (DPI) or apocynin, or scavenging H₂O₂ by catalase overexpression markedly impaired cardiac regeneration while exogenous H₂O₂ rescued the inhibitory effects of DPI on cardiac regeneration, indicating that H₂O₂ is an essential and sufficient signal in this process. Mechanistically, elevated H₂O₂ destabilized the redox-sensitive phosphatase Dusp6 and hence increased the phosphorylation of Erk1/2. The Dusp6 inhibitor BCI achieved similar pro-regenerative effects while transgenic overexpression of dusp6 impaired cardiac regeneration. H₂O₂ plays a dual role in recruiting immune cells and promoting heart regeneration through two relatively independent pathways. We conclude that H₂O₂ potentially generated from Duox/Nox2 promotes heart regeneration in zebrafish by unleashing MAP kinase signaling through a derepression mechanism involving Dusp6.     

Testosterone Stimulates Duox1 Activity through GPRC6A in Skin Keratinocytes

Testosterone is an endocrine hormone with functions in reproductive organs, anabolic events, and skin homeostasis. We report here that GPRC6A serves as a sensor and mediator of the rapid action of testosterone in epidermal keratinocytes. The silencing of GPRC6A inhibited testosterone-induced intracellular calcium ([Ca²⁺]_i) mobilization and H₂O₂ generation. These results indicated that a testosterone-GPRC6A complex is required for activation of G_q protein, IP₃ generation, and [Ca²⁺]_i mobilization, leading to Duox1 activation. H₂O₂ generation by testosterone stimulated the apoptosis of keratinocytes through the activation of caspase-3. The application of testosterone into three-dimensional skin equivalents increased the apoptosis of keratinocytes between the granular and stratified corneum layers. These results support an understanding of the molecular mechanism of testosterone-dependent apoptosis in which testosterone stimulates H₂O₂ generation through the activation of Duox1.     

Silencing of miR-23a attenuates hydrogen peroxide (H2O2) induced oxidative damages in ARPE-19 cells by upregulating GLS1: an in vitro study

BackgroundOxidative damages contributes to age-related macular degeneration (AMD) caused vision blindness, but the molecular mechanisms are still largely unknown.ObjectivesThis study managed to investigate this issue by conducting in vitro experiments.MethodsOxidative stress were evaluated by L-012 dye, DHE staining and MDA assay. CCK-8 and colony formation assay were conducted to examine cell proliferation. Cell death was evaluated by trypan blue staining and Annexin V-FITC/PI double staining method through flow cytometry (FCM). The binding sites of miR-23a and GLS1 mRNA were predicted by online miRDB database and validated by dual-luciferase reporter gene system. Real-Time qPCR for miR-23a levels and Western Blot for protein expressions.ResultsThe retinal pigment epithelial (RPE) cells (ARPE-19) were subjected to hydrogen peroxide (H₂O₂) stimulation to simulate AMD progression in vitro, and we identified a novel miR-23a/glutaminase-1 (GLS1) pathway that regulated H₂O₂ induced oxidative damages in ARPE-19 cells. Mechanistically, H₂O₂ induced oxidative stress, inhibited cell proliferation and induced cell death in ARPE-19 cells in a dose- and time-dependent manner. Also, H₂O₂ stimulation hindered cell invasion, migration and glutamine uptake in ARPE-19 cells. Interestingly, we proved that H₂O₂ increased miR-23a levels, while downregulated glutaminase-1 (GLS1) in ARPE-19 cells, and miR-23a targeted 3′ untranslated region (3′UTR) of GLS1 mRNA for GLS1 degradation. Finally, our data suggested that silencing miR-23a upregulated GLS1 to reverse the detrimental effects of H₂O₂ treatment on ARPE-19 cells.ConclusionsIn general, analysis of the data suggested that miR-23a ablation upregulated GLS1 to attenuate H₂O₂ stimulation induced oxidative damages in ARPE-19 cells in vitro, and this study broadened our knowledge in this field, which might help to provide novel theranostic signatures for AMD.     

Presence of presenilin 1/2 affects the invasion and replication of Salmonella typhimurium

In this study, we used four different cell lines, with or without presenilin-1 or -2, to investigate the hypothesis that the presence of presenilin, the most prominently mutated gene in Alzheimer’s patients, affects the infection rate of host cells by Salmonella. The invasion and replication of Salmonella in presenilin 1/2-deficient cells were significantly lower than those in presenilin 1/2-expressing cells. Among several presenilin-interacting proteins, the expression of filamin-A in presenilin 1/2-deficient cells was significantly lower than in presenilin 1/2-expressing cells. However, Salmonella infection of filamin-A-deficient M2 cells did not significantly differ from infection of filamin-A-containing A7 cells, ruling out the possibility that filamin-A is a major protein inhibiting Salmonella invasion and replication. It is of interest to note that Hes-1 expression, a downstream target of Notch signaling pathway, was significantly decreased by Salmonella infection. Our results demonstrate that the presence of presenilins affects the invasion and replication processes of Salmonella.     

E. coli Nissle 1917 Affects Salmonella adhesion to porcine intestinal epithelial cells

Background

The probiotic Escherichia coli strain Nissle 1917 (EcN) has been shown to interfere in a human in vitro model with the invasion of several bacterial pathogens into epithelial cells, but the underlying molecular mechanisms are not known.

Methodology/Principal Findings

In this study, we investigated the inhibitory effects of EcN on Salmonella Typhimurium invasion of porcine intestinal epithelial cells, focusing on EcN effects on the various stages of Salmonella infection including intracellular and extracellular Salmonella growth rates, virulence gene regulation, and adhesion. We show that EcN affects the initial Salmonella invasion steps by modulating Salmonella virulence gene regulation and Salmonella SiiE-mediated adhesion, but not extra- and intracellular Salmonella growth. However, the inhibitory activity of EcN against Salmonella invasion always correlated with EcN adhesion capacities. EcN mutants defective in the expression of F1C fimbriae and flagellae were less adherent and less inhibitory toward Salmonella invasion. Another E. coli strain expressing F1C fimbriae was also adherent to IPEC-J2 cells, and was similarly inhibitory against Salmonella invasion like EcN.

Conclusions

We propose that EcN affects Salmonella adhesion through secretory components. This mechanism appears to be common to many E. coli strains, with strong adherence being a prerequisite for an effective reduction of SiiE-mediated Salmonella adhesion.     

Dependence of leukemic cell proliferation and survival on H2O2 and l-arginine

The proliferation and/or survival of a variety of cells is dependent on cellular hydrogen peroxide (H₂O₂) production. We tested whether this was true of leukemic cells, using cell lines from leukemic patients (CEM, 697, Mn-60, and Tanoue). We found that addition of catalase inhibited proliferation of all cell lines and induced death in two. However, this turned out to be due to arginase contamination of the catalase. Pure arginase inhibited cell proliferation and survival, which was reversible by adding l-arginine, demonstrating the l-arginine dependency of these cells. The glutathione peroxidase mimetic ebselen killed the cells by a novel, rapid form of death, preceded by cell blebbing and prevented by N-acetylcysteine, suggesting toxicity is not due to ebselen's antioxidant activity. Addition of N-acetylcysteine to remove endogenous H₂O₂ stimulated survival and proliferation, suggesting that basal levels of H₂O₂ promoted cell death. Consistent with this, leukemic cell death was induced by adding as little as 5 μM H₂O₂. Ascorbic acid, even at 100 μM, induced death through H₂O₂ production. Thus H₂O₂ does not promote proliferation and survival, rather the opposite, and previous literature may have misinterpreted the effects of antioxidants. Arginase, H₂O₂, ascorbic acid, and ebselen might be useful in the treatment of leukemia.