In vitro killing of microfilariae of Brugia pahangi and Brugia malayi by eosinophil granule proteins

Eosinophil infiltration and degranulation around the tissue-invasive stages of several species of helminths have been observed. Release of eosinophil granule contents upon the worms is supported by localization of two of the major granule proteins, major basic protein (MBP) and eosinophil peroxidase (EPO), on and around species of trematodes, nematodes, and cestodes. In the case of filarial worms, MBP is deposited on degenerating microfilariae (mf) of Onchocerca volvulus. Here, we performed in vitro assays of the toxicity of four purified eosinophil granule proteins, namely, MBP, EPO, eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN), for the mf of Brugia pahangi and Brugia malayi. MBP, ECP, and EDN killed these worms in a dose-related manner although relatively high concentrations of EDN were necessary. EPO, in the presence of a H2O2-generating system and a halide, was the most potent toxin on a molar basis; here, the most potent halide was I- followed by Br- and Cl-. Surprisingly, EPO in the absence of H2O2 killed mf at concentrations comparable to those required for MBP and ECP. The toxicity of EPO + H2O2 + halide was inhibited by heparin, catalase, or 1% BSA, whereas the toxicity of EPO alone was inhibited only by heparin. Heparin also inhibited killing by both MBP and ECP. Despite the homology of ECP with certain RNases, placental RNasin, an RNase inhibitor, was unable to inhibit ECP-mediated toxicity. These results indicate that all of the eosinophil granule proteins are toxic to mf and they support the hypothesis that eosinophil degranulation causes death of mf in vivo.     

Peroxidase-mediated toxicity to schistosomula of Schistosoma mansoni

Guinea pig eosinophil peroxidase (EPO) was capable of killing schistosomula of Schistosoma mansoni in vitro when combined with hydrogen peroxide and a halide. Killing was measured by 51Cr release, by microscopic evaluation of viability, and by reinfection experiments in mice. Parasite killing was dependent on each component of the EPO-H2O2-halide system, was completely inhibited by catalase and azide, and was partially inhibited by cyanide. The EPO-mediated system required 10(-4) M H2O2 and 10(-4) M iodide at pH 7.0, and the schistosomula were killed with exposure to this system of less than 30 min at 37 degrees C. At pH 6.0, the EPO-mediated system showed significant cidal activity with 10(-6) M iodide. Canine neutrophil peroxidase (myeloperoxidase [MPO]) was also able to kill schistosomula in vitro in the presence of 10(-4) M H2O2 and 10(-4) iodide at pH 7.0 and pH 6.0. Physiologic concentrations of chloride (0.1 M) could substitute for iodide at pH 7.0 and pH 6.0 as the halide cofactor; however, at pH 7.0, a higher concentration of enzyme was required. These findings with isolated enzyme systems are compatible with a role for peroxidase in the host defense against schistosomula.     

Parasiticidal activity of bovine lactoperoxidase against Toxoplasma gondii.

Toxoplasma gondii is an obligatory intracellular parasitic protozoan transmitted via the ingestion of raw, infected meat that causes congenital infections. In a cell-free environment, virulent Toxoplasma was strikingly resistant to H2O2. The activity of H2O2 or H2O2 generated by glucose-glucose oxidase against the resistant tachyzoite stage of pathogenic T. gondii was enhanced by adding KI and bovine lactoperoxidase (bLPO), referred to here as the bLPO system. Replacing bLPO (heme content, 90%) with recombinant bLPO (heme content, 6%) did not enhance the parasiticidal activity with KI and H2O2. These results indicated that heme contributed to the enzyme activity and resulted in the killing of tachyzoites of T. gondii. Tachyzoites treated with the bLPO system also lost the ability to penetrate the mouse fibroblast cell line (NIH/3T3), and could be killed intracellularly after exposure by bLPO to a mouse macrophage cell line (J774A.1). These findings suggested that toxicity was mediated through small amounts of H2O2 generated by phagocytic events in naive macrophages, and by the peroxidative activity of bLPO. Our observations suggest that the bLPO system could help prevent the development of Toxoplasmosis in humans after ingesting raw, infected meat.     

Role of polymorphonuclear cells in Chagas' disease. I. Uptake and mechanisms of destruction of intracellular (amastigote) forms of Trypanosoma cruzi by human neutrophils

The ability of human polymorphonuclear cells (PMN) to take up and destroy intracellular forms of Trypanosoma cruzi (AMA) was investigated as a part of our efforts to elucidate the mechanisms of clearing of these parasites from infected tissues. PMN were found to take up AMA and destroyed parasites were seen after 30 min of cell-parasite interaction. Under our experimental conditions, the rate of uptake of AMA by PMN was maximal during the first 30 min of interaction. AMA were found to be located and destroyed inside the phagolysosomal vacuoles of PMN. The parasite was never found outside these vacuoles despite electron microscopic examination of numerous preparations derived from several experiments. Intracellular destruction of AMA by PMN was visible by electron microscopy and could be monitored by measuring the release of 3H-labeled substances by PMN that had ingested radiolabeled AMA. PMN incubated after removal of unbound parasites destroyed over 90% of the ingested organisms within 3 hr and close to 99% after 12 hr. In cellfree systems, 44% of the AMA were destroyed in the presence of 10(-4) M H2O2 and all of the parasites died at 10(-3) M. Addition of lactoperoxidase and iodide resulted in 100% killing at 10(-5) M H2O2. These mechanisms appeared to be involved in the lysis of AMA by PMN since both H2O2 and peroxidase activity were demonstrated to be present in PMN vacuoles containing the parasite. Addition of NaN3, KCN (inhibitors of myeloperoxidase activity) or catalase (to decompose H2O2) caused a marked reduction in the extent of AMA killing by PMN. Xanthine oxidase was toxic for the AMA in the presence of acetaldehyde. This microbicidal activity was inhibited by catalase but not by heat-inactivated catalase or by reagents that scavenge the intermediate products of reduction of molecular oxygen, O - X 2, X OH, and 1O2. These results suggest that PMN have the potential of clearing AMA liberated in infected chagasic tissues and that parasite killing within the phagolysosomal vacuoles is mediated by myeloperoxidase activity and H2O2.     

Eosinophil peroxidase-mediated inactivation of leukotrienes B4, C4, and D4

The slow-reacting substance (SRS) bioactivity of leukotrienes C4 (LTC4) and D4 (LTD4) was rapidly decreased by incubation with eosinophil peroxidase (EPO), H2O2, and iodide, bromide, or to a lesser degree, chloride, LTB4 chemotactic activity was also decreased by the EPO-H2-H2-halide system, although at a slower rate. Myeloperoxidase could substitute for EPO in these reactions. Leukotriene inactivation was greatly decreased or abolished by deletion of any of the components of the system or by the addition of the hemeprotein inhibitors, azide, cyanide, or aminotriazole, indicating a requirement for peroxidase. The H2O2 concentration employed in the above studies was 10(-4) M. H2O2 at higher concentrations (5 x 10(-4) to 10(-2) M) inactivated LTC4 and LTD4 in the absence of EPO and a halide but had no effect on the chemotactic activity of LTB4. We have previously shown that horse eosinophils stimulated with the calcium ionophore A23187 generate SRS. In the present study, eosinophils stimulated in this way were found to release extracellularly both H2O2 and EPO. Incubation of eosinophils with azide that inhibits EPO, and catalase that degrades H2O2, significantly increased the amount of SRS activity detected in the extracellular medium after A23187 stimulation. These findings suggests eosinophils may play an important modulating role in hypersensitivity reactions both by the production of leukotrienes and by their inactivation through the release of H2O2 and EPO.     

Diffusion of extracellular hydrogen peroxide into intracellular compartments of human neutrophils. Studies utilizing the inactivation of myeloperoxidase by hydrogen peroxide and azide

It is well known that catalase is transformed to nitric oxide-Fe2+-catalase by hydrogen peroxide (H2O2) plus azide. In this report, we show that myeloperoxidase is also inactivated by H2O2 plus azide. Utilizing this system, we studied the presence and source of intracellular H2O2 generated by activated neutrophils. Stimulation of neutrophils with phorbol myristate acetate (PMA, 100 ng/ml) plus azide (5 mM) for 30 min completely inactivated intragranular myeloperoxidase and reduced cytosolic catalase to 35% of resting cells. This intracellular inactivation of heme enzymes did not occur in normal neutrophils incubated with either PMA or azide alone or in neutrophils from patients with chronic granulomatous disease (CDG) which cannot produce H2O2 in response to PMA. Incubation of neutrophils with azide and a H2O2 generating system (glucose-glucose oxidase) inactivated 41% of neutrophil myeloperoxidase. Glutathione-glutathione peroxidase (GSH-GSH peroxidase), an extracellular H2O2 scavenger, totally protected neutrophil myeloperoxidase from inactivation by azide plus glucose-glucose oxidase. In addition, when a mixture of normal and CGD cells was stimulated with PMA in the presence of azide, 90% of the myeloperoxidase in CGD neutrophils was inactivated. Therefore, H2O2 released extracellularly from activated neutrophils can diffuse into cells. In contrast, myeloperoxidase in normal polymorphonuclear leukocytes stimulated with PMA in the presence of azide and GSH-GSH peroxidase was 75% inactivated. Thus, the results indicate that a GSH-GSH peroxidase-insensitive pool of H2O2 is also generated, presumably at the plasma membrane, and this pool of H2O2 can undergo direct internal diffusion to inactivate myeloperoxidase.     

Catalase and superoxide dismutase in Escherichia coli

We assessed the roles of intrabacterial catalase and superoxide dismutase in the resistance of Escherichia coli to killing by neutrophils. E. coli in which the synthesis of superoxide dismutase and catalase were induced by paraquat 10-fold and 5-fold, respectively, did not resist killing by neutrophils. When bacteria were allowed to recover from the toxicity of paraquat for 1 h on ice and for 30 min at 37 degrees C, they still failed to resist killing by neutrophils. Induction of the synthesis of catalase 9-fold by growth in the presence of phenazine methosulfate did not render E. coli resistant to killing by either neutrophils or by H2O2 itself. The lack of protection by intrabacterial catalase from killing by neutrophils could not be attributed to an impermeable bacterial membrane; the evolution of O2 from H2O2 was no less rapid in suspensions of E. coli than in lysates. The failure of intrabacterial catalase or superoxide dismutase to protect bacteria from killing by neutrophils might indicate either that the flux of O-2 and H2O2 in the phagosome is too great for the intrabacterial enzymes to alter or that the site of injury is at the bacterial surface.     

Down-regulation of human natural killer activity against tumors by the neutrophil myeloperoxidase system and hydrogen peroxide

There is growing evidence that natural killer (NK) cells play an important role in immune surveillance against tumors and certain infections. The coexistence of activated neutrophils with lymphocytes in tumor masses and inflammatory tissues suggests the possibility of interaction between secreted neutrophil products and nearby lymphocytes. We examined the susceptibility of lymphocyte NK activity to oxidative injury by the neutrophil myeloperoxidase (MPO) system and H2O2 with the use of a cellfree model system. Exposure of human mononuclear leukocytes (MNL) to MPO, an H2O2-generating system (glucose + glucose oxidase), and a halide (C1- or I-) resulted in marked suppression of MNL-NK activity, as measured by 51Cr release from K562 tumor targets (p less than 0.001). This suppression was dependent on the presence and activity of each system component and was blocked by azide and catalase, but not by heated catalase. In spite of the marked functional suppression of NK activity, MNL viability was more than 95% and target binding frequency was not affected. NK suppression was reversible after 24 hr in culture. The mechanism of suppression was dependent on the amount and rate of H2O2 delivered, and on MNL number. MPO was essential when H2O2 flux was low or when MNL numbers were high. As H2O2 flux increased or MNL numbers decreased, NK suppression gradually became MPO-independent and was mediated by H2O2 alone. The ability of the MPO system to compromise lymphocyte NK function may explain the in vitro inhibition of NK activity of mixed cell populations by the tumor promoter phorbol esters, because these agents are potent stimulants for neutrophil secretion of MPO and H2O2. This study may also provide a possible mechanism for the reported in situ NK activity suppression by adherent phagocytic cells during carcinogenesis in both humans and animals.     

The peptidoglycan-associated lipoprotein OprL helps protect a Pseudomonas aeruginosa mutant devoid of the transactivator OxyR from hydrogen peroxide-mediated killing during planktonic and biofilm culture

OxyR controls H(2)O(2)-dependent gene expression in Pseudomonas aeruginosa. Without OxyR, diluted (<10(7)/ml) organisms are easily killed by micromolar H(2)O(2). The goal of this study was to define proteins that contribute to oxyR mutant survival in the presence of H(2)O(2). We identified proteins in an oxyR mutant that were oxidized by using 2,4-dinitrophenylhydrazine for protein carbonyl detection, followed by identification using a two-dimensional gel/matrix-assisted laser desorption ionization-time of flight approach. Among these was the peptidoglycan-associated lipoprotein, OprL. A double oxyR oprL mutant was constructed and was found to be more sensitive to H(2)O(2) than the oxyR mutant. Provision of the OxyR-regulated alkyl hydroperoxide reductase, AhpCF, but not AhpB or the catalase, KatB, helped protect this strain against H(2)O(2). Given the sensitivity of oxyR oprL bacteria to planktonic H(2)O(2), we next tested the hypothesis that the biofilm mode of growth might protect such organisms from H(2)O(2)-mediated killing. Surprisingly, biofilm-grown oxyR oprL mutants, which (in contrast to planktonic cells) possessed no differences in catalase activity compared to the oxyR mutant, were sensitive to killing by as little as 0.5 mM H(2)O(2). Transmission electron microscopy studies revealed that the integrity of both cytoplasmic and outer membranes of oxyR and oxyR oprL mutants were compromised. These studies suggest that sensitivity to the important physiological oxidant H(2)O(2) in the exquisitely sensitive oxyR mutant bacteria is based not only upon the presence and location of OxyR-controlled antioxidant enzymes such as AhpCF but also on structural reinforcement by the peptidoglycan-associated lipoprotein OprL, especially during growth in biofilms.     

Production of H2O2 by bovine blood and milk polymorphonuclear leucocytes

The ability of bovine polymorphonuclear leucocytes (PMN) to release H2O2 was investigated. Resting PMN suspended in buffer released only small amounts of H2O2 which was appreciably increased during phagocytosis of heat killed coliforms. However, in the presence of bovine serum (BS), foetal calf serum (FCS) and milk whey (MW) no increase of H2O2 could be detected unless sodium azide (NaN2) was added. It appears that the enzyme content of these fluids (catalase and lactoperoxidase) consumed the released H2O2 and NaN2, which inactivates these enzymes, abolished this interference. Live organisms required BS or MW both for phagocytosis and for H2O2 production. Bovine IgG2 and, to a lesser extent, IgG1 but not SIgA or IgM stimulated the release of H2O2 independently of phagocytosis; this indicates the presence of receptors specific for IgG2 and IgG1 on the cell surface. Ingestion of casein micelles triggered the greatest burst of H2O2 production by cells suspended in buffer. In general, PMN isolated from blood were more active than cells isolated from milk. Since the extracellular release of H2O2 reflects the intracellular level of H2O2, the lower metabolic activity of milk PMN may contribute to the lesser intracellular bactericidal activity of milk leucocytes. The possibility that the release of H2O2 may activate extracellularly the lactoperoxidase system, known to be bactericidal in milk, is discussed.     

Human serum catalase decreases endothelial cell injury from hydrogen peroxide.

Serum from normal human subjects contained variable amounts of catalase activity, which was inhibitable by heat, azide, trichloroacetic acid (TCA), or aminotriazole treatment. Serum also decreased hydrogen peroxide (H2O2) concentrations in vitro and H2O2-mediated injury to cultured endothelial cells. By comparison, heat-, azide-, TCA-, or aminotriazole-treated serum neither decreased H2O2 concentrations in vitro nor reduced H2O2-mediated damage to endothelial cells. We conclude that serum catalase activity can alter H2O2-dependent reactions. We speculate that variations in serum catalase activity may alter individual susceptibility to oxidant-mediated vascular disease or be a factor when added to test systems in vitro.     

Oxidative mechanisms of toxicity of low-intensity near-UV light in Salmonella typhimurium.

The exposure of Salmonella typhimurium to environmentally relevant near-UV light stress has been studied by the use of a low-intensity, broad-band light source. The exposure of cells to such a light source rapidly induced a growth delay; after continuous exposure for 3 to 4 h, cells began to die at a rapid rate. The oxidative defense regulon controlled by the oxyR gene was involved in protecting cells from being killed by near-UV light. This killing may be potentiated by the overexpression of near-UV-absorbing proteins. These results are consistent with near-UV toxicity involving the absorption of light by endogenous photosensitizers, leading to the production of active oxygen species. We have shown, however, that one such species, H2O2, is not a major photoproduct involved in killing by near-UV light. Strains lacking alkyl hydroperoxide reductase were more sensitive to near-UV light, indicating that such hydroperoxides may be photoproducts. Near-UV exposure induced sensitivity to high salt levels, indicating that membranes may be a target of near-UV toxicity and a possible source of alkyl hydroperoxides. The demonstration of the inactivation of the heme-containing protein catalase indicates that direct destruction of UV-absorbing macromolecules could be another factor in near-UV toxicity. Cells which have been exposed to near-UV light for long, but sublethal, periods of time (up to 4 h) can recover and resume growth if the UV exposure is stopped but become progressively more sensitive to further stresses, such as H2O2. This result indicates that cells gradually accumulated damage during near-UV exposure until toxic levels were reached.     

Mechanisms of the killing of cultured hepatocytes by hydrogen peroxide

Mechanisms of H2O2-induced cell injury were explored in primary cultures of rat hepatocytes. Cells prepared from male rats and cultured for 1 day prior to treatment were killed by H2O2 either added directly to the medium at 0.25-2 mM or generated in situ by glucose oxidase (0.25-2 U/ml) or xanthine oxidase (20-120 mM/ml) and 2 mM xanthine. Catalase protected the cells in each case. Lipid peroxidation as measured by the accumulation of malondialdehyde (MDA) preceded the cell death due to H2O2 added directly to the cultures or generated in the medium. The antioxidants N,N'-diphenyl-p-phenylenediamine (DPPD) and promethazine prevented the accumulation of MDA in both cases and protected the cells treated with H2O2 directly. DPPD and promethazine did not react directly with H2O2. Other antioxidants including butylated hydroxytoluene, vitamin E, and N-propylgallate had varied protective activity against the addition of H2O2 in proportion to their ability to reduce MDA accumulation. In glucose oxidase-treated cultures, DPPD and promethazine prevented the cell killing during the first hour but failed to protect between 1 and 3 h despite prevention of lipid peroxidation. The cell killing between 1 and 3 h in the presence of DPPD was prevented by catalase indicating its dependence upon continued generation of H2O2. Further addition of H2O2 in the presence of DPPD also increased the number of dead cells without lipid peroxidation. The data are consistent with at least two mechanisms of hepatocyte killing by H2O2. The first pathway is prevented by the antioxidants DPPD and promethazine and is very likely related to the peroxidation of membrane phospholipids. The second is independent of lipid peroxidation yet dependent upon the continued presence of H2O2.     

Human phagocytic cell responses to Mycobacterium leprae and Mycobacterium bovis Bacillus Calmette-Guérin. An in vitro comparison of leprosy vaccine components

Components of current vaccines for Hansen's disease include Mycobacterium bovis Bacillus Calmette-Guérin (BCG) and killed Mycobacterium leprae. BCG infections in humans are rare and most often occur in immune-compromised individuals. M. leprae on the other hand, although not causing clinical disease in most exposed individuals, is capable of infecting and replicating within mononuclear phagocytes. Lymphocytes from patients with the lepromatous form of Hansen's disease exhibit defective lymphokine production when challenged in vitro with M. leprae. This may result in inefficient mononuclear phagocyte activation for oxidative killing. To study the ability of normal phagocytes to ingest and respond oxidatively to BCG and M. leprae, we measured phagocytic cell O2- release and fluorescent oxidative product formation and visually confirmed the ingestion of the organisms. BCG stimulated a vigorous O2- generation in neutrophils and monocytes and flow cytometric oxidative product generation by neutrophils occurred in the majority of cells. M. leprae, stimulated a weak but significant O2- release requiring a high concentration of organisms and long exposure. By flow cytometric analysis, most neutrophils were able to respond to both organisms with the generation of fluorescent oxidative products. Neutrophil oxidative responses to M. leprae were substantially less than responses seen from neutrophils exposed to BCG. By microscopic examination of neutrophils phagocytizing FITC-labeled bacteria, it was shown that both M. leprae and BCG were slowly ingested but that more BCG appeared to be associated with the cell membrane of more of the cells. When phagocytic cells were incubated with BCG and M. leprae for 30 min and subsequently examined by electron microscopy, few organisms were seen in either neutrophils or monocytes. This suggests that BCG are easily recognized and slowly ingested by normal phagocytic cells, the majority of which respond with a strong oxidative burst. M. leprae appeared to only weakly stimulate phagocyte oxidative responses and were also slowly phagocytized.     

Killing of Schistosoma mansoni sporocysts by hemocytes from resistant Biomphalaria glabrata: role of reactive oxygen species

The fate of Schistosoma mansoni (Trematoda) sporocysts in its molluscan host Biomphalaria glabrata (Gastropoda) is determined by circulating phagocytes (hemocytes). When the parasite invades a resistant snail, it is attacked and destroyed by hemocytes, whereas in a susceptible host it remains unaffected. We used 3 inbred strains of B. glabrata: 13-16-R1 and 10-R2, which are resistant to the PR-1 strain of S. mansoni, and M-line Oregon (MO), which is susceptible to PR-1. In an in vitro killing assay using plasma-free hemocytes from these strains, the rate of parasite killing corresponded closely to the rate by which S. mansoni sporocysts are killed in vivo. Hemocytes from resistant snails killed more than 80% of S. mansoni sporocysts within 48 hr, whereas sporocyst mortality in the presence of hemocytes from susceptible snails was <10%. Using this in vitro assay, we assessed the involvement of reactive oxygen species (ROS) produced by resistant hemocytes, during killing of S. mansoni sporocysts. Inhibition of NADPH oxidase significantly reduced sporocyst killing by 13-16-R1 hemocytes, indicating that ROS play an important role in normal killing. Reduction of hydrogen peroxide (H2O2) by including catalase in the killing assay increased parasite viability. Reduction of superoxide (O2-), however, by addition of superoxide dismutase or scavenging of hydroxyl radicals (*OH) and hypochlorous acid (HOCl) by addition of hypotaurine did not alter the rate of sporocyst killing by resistant hemocytes. We conclude that H2O2 is the ROS mainly responsible for killing.     

Toxicity of the sulfhydryl-containing radioprotector dithiothreitol

The toxicity of the sulfhydryl-containing radioprotective agent dithiothreitol (DTT) has been studied using Chinese hamster V79 cells growing in monolayer in minimal essential medium containing 10% fetal calf serum. DTT at low concentrations (between 0.4 and 1.0 mM) caused cell killing, but higher concentrations (above 2 mM) or lower concentrations (0.1 mM) did not. This DTT-induced toxicity was prevented by catalase, glutathione, the use of serum-free medium, or lowering incubation temperature; was slightly decreased by dimethyl sulfoxide; and was enhanced by some metal chelators but prevented by desferal, an iron chelator. Experiments involving simultaneous exposure of cells to DTT and H2O2 showed that low concentrations of DTT enhanced H2O2-induced toxicity, but high concentrations of DTT prevented the H2O2 toxicity. These results are consistent with the proposal that toxicity results from autoxidation of DTT to produce H2O2, which in turn reacts via the metal-catalyzed Fenton reaction to produce the ultimate toxin, .OH radicals, although chemical studies show that rates of autoxidation of various sulfhydryl compounds do not correlate with the observed toxicity.     

Mechanisms of mutagenicity and toxicity of sodium selenite (Na2SeO3) in Salmonella typhimurium

The mechanisms of selenite toxicity and mutagenicity in S. typhimurium have been characterized. In contrast to previous reports, selenite toxicity was shown not to involve nonspecific incorporation into protein via the sulfur metabolic pathways. Selenite toxicity was, however, shown to involve its ability to act as an oxidizing agent, primarily through reactions with sulfhydryls. Strains which lack glutathione (GSH) are more sensitive to killing by sulfhydryl reagents. The selenite sensitivity of such a mutant was a biphasic phenomenon. The mutant was much more sensitive than a strain which contained GSH at lower selenite concentrations whereas, at higher concentrations, the mutant was much more resistant to selenite. The mechanism of selenite toxicity at lower concentrations in this mutant thus appeared to involve damage to intracellular sulfhydryls. The sensitization to higher doses of selenite by GSH could be explained by the generation of toxic oxygen species. The in vitro reactions of selenite with both cysteine and GSH readily produced H2O2 and O2-. A S. typhimurium strain which overproduces superoxide dismutase (SOD) and catalase was more resistant to high concentrations of selenite, but not killing by the lower doses. Pretreatment of cells with a nonlethal dose of selenite induced the synthesis of proteins which protected the cells from killing by H2O2 or high doses of selenite. Selenite was also a mutagen in the tester strain TA104, in which a number of other oxidizing agents have also been found to be mutagens. These results were consistent with a model in which the reactions of selenite and intracellular thiols with concomitant production of active oxygen species are the primary causal agents of selenite mutagenicity and toxicity in S. typhimurium.     

Chemotactic factor inactivation by stimulated human neutrophils mediated by myeloperoxidase-catalyzed methionine oxidation

Leukocyte chemoattractants were inactivated when exposed to human neutrophils and either ingestible particles or phorbol esters. Loss of biologic activity was time- and temperature-dependent, required physiologic concentrations of viable neutrophils and a halide, and was inhibited by azide or catalase. Neutrophils from patients with either hereditary myeloperoxidase deficiency or chronic granulomatous disease failed to inactivate the chemoattractants unless purified myeloperoxidase or H2O2, respectively, was added. Susceptibility to inactivation by neutrophils correlated with the presence of methionine in the attractant. Loss of chemotactic activity was blocked by low concentrations of methionine and by higher concentrations of other reducing agents, but was unaffected by oxidized methionine. Paper chromatography demonstrated that exposure of a formyl-methionyl peptide chemotactic factor to either the cellfree myeloperoxidase system or stimulated neutrophils resulted in its conversion to a molecular species whose location in the chromatographs was identical to that of the peptide containing oxidized methionine. Thus, stimulated human neutrophils inactivate peptide chemoattractants by secretion of myeloperoxidase and H2O2, which combine with halides to form oxidants that react with a critical methionine residue. We suggest that myeloperoxidase-catalyzed oxidation of thioethers may constitute an inflammatory control mechanism as well as a general means of modifying the functional properties of biologic mediators.     

Microbial resistance in relation to catalase activity to oxidative stress induced by photolysis of hydrogen peroxide

The purpose of the present study was to evaluate the mechanism of microbial resistance to oxidative stress induced by photolysis of hydrogen peroxide (H(2)O(2)) in relation to microbial catalase activity. In microbicidal tests, Staphylococcus aureus and Candida albicans were killed and this was accompanied by production of hydroxyl radicals. C. albicans was more resistant to hydroxyl radicals generated by photolysis of H(2)O(2) than was S. aureus. A catalase activity assay demonstrated that C. albicans had stronger catalase activity; accordingly, catalase activity could be one of the reasons for the resistance of the fungus to photolysis of H(2)O(2). Indeed, it was demonstrated that C. albicans with strong catalase activity was more resistant to photolysis of H(2)O(2) than that with weak catalase activity. Kinetic analysis using a modified Lineweaver-Burk plot also demonstrated that the microorganisms reacted directly with hydroxyl radicals and that this was accompanied by decomposition of H(2)O(2). The results of the present study suggest that the microbicidal effects of hydroxyl radicals generated by photolysis of H(2)O(2) can be alleviated by decomposition of H(2)O(2) by catalase in microorganisms.     

Mutagenesis and stress responses induced in Escherichia coli by hydrogen peroxide

Killing of Escherichia coli by hydrogen peroxide proceeds by two modes. Mode one killing appears to be due to DNA damage, has a maximum near 1 to 3 mM H2O2, and requires active metabolism during exposure. Mode two killing is due to uncharacterized damage, occurs in the absence of metabolism, and exhibits a classical multiple-order dose-response curve up to at least 50 mM H2O2 (J. A. Imlay and S. Linn, J. Bacteriol. 166:519-527, 1986). H2O2 induces the SOS response in proportion to the degree of killing by the mode one pathway, i.e., induction is maximal after exposure to 1 to 3 mM H2O2. Mutant strains that cannot induce the SOS regulon are hypersensitive to peroxide. Analysis of the sensitivities of mutants that are deficient in individual SOS-regulated functions suggested that the SOS-mediated protection is due to the enhanced synthesis of recA protein, which is rate limiting for recombinational DNA repair. Specifically, strains wholly blocked in both SOS induction and DNA recombination were no more sensitive than mutants that are blocked in only one of these two functions, and strains carrying mutations in uvrA, -B, -C, or -D, sfiA, umuC or -D, ssb, or dinA, -B, -D, -F, -G, -H, -I, or -J were not abnormally sensitive to killing by H2O2. After exposure to H2O2, mutagenesis and filamentation also occurred with the dose response characteristic of SOS induction and mode one killing, but these responses were not dependent on the lexA-regulated umuC mutagenesis or sfiA filamentation functions, respectively. Exposure of E. coli to H2O2 also resulted in the induction of functions under control of the oxyR regulon that enhance the scavenging of active oxygen species, thereby reducing the sensitivity to H2O2. Catalase levels increased 10-fold during this induction, and katE katG mutants, which totally lack catalase, while not abnormally sensitive to killing by H2O2 in the naive state, did not exhibit the induced protective response. Protection equal to that observed during oxyR induction could be achieved by the addition of catalase to cultures of naive cells in an amount equivalent to that induced by the oxyR response. Thus, the induction of catalase is necessary and sufficient for the observed oxyR-directed resistance to killing by H2O2. Although superoxide dismutase appeared to be uninvolved in this enhanced protective response, sodA sodB mutants, which totally lack superoxide dismutase, were especially sensitive to mode one killing by H2O2 in the naive state. gshB mutants, which lack glutathione, were not abnormally sensitive to killing by H2O2.