Alpha 1-antitrypsin variants produced by recombinant DNA: differences in elastase inhibitory activity and resistance to oxidant agents

Inherited or "acquired" deficiency of alpha 1-antitrypsin (believed to be the cause of pulmonary emphysema) will probably be treated in the future by replacement with alpha 1-antitrypsin purified from human plasma or produced by recombinant DNA, which seems promising because it permits site-specific mutagenesis in the oxidizable active site of the normal human alpha 1-antitrypsin. The aim of this in-vitro study was to investigate the elastase inhibitory activity and the resistance to oxidizing agents of normal human alpha 1-antitrypsin, a recombinant yeast-produced variant (VAL 358) and a recombinant E. coli-produced variant (LEU 358). The inhibitors were exposed to chemical oxidants (NCS, H2O2, xanthine/xanthine oxidase, chloramine-T) and to PMA-activated neutrophils. The elastase inhibitory activity was assayed on porcine pancreatic elastase and neutrophil elastase. Normal alpha 1-antitrypsin and VAL 358 variant were good inhibitors of both elastases. LEU 358 variant was the best inhibitor for neutrophil elastase, but it poorly inhibited the porcine pancreatic elastase. Normal alpha 1-antitrypsin was affected by all oxidants; both variants were almost totally resistant to chemical oxidants and to activated neutrophils. We conclude that recombinant alpha 1-antitrypsin variants differ in their elastase inhibitory activity and offer increased resistance to oxidant agents.     

Antioxidant properties of pyrrolidine dithiocarbamate and its protection against Cr(VI)-induced DNA strand breakage

Pyrrolidine dithiocarbamate (PDTC) is considered an antioxidant and is frequently used to study the role of free radical reactions in various biological processes and against free radical-induced cellular injuries. However, its antioxidant properties are not characterized. In this study, electron spin resonance (ESR) was used to investigate the antioxidant potential of PDTC with hydroxyl radical (*OH) and superoxide anion radicals (O2*-). The Fenton reaction [Fe(II) + H2O2 --> Fe(II) + *OH + OH-)] and xanthine and xanthine oxidase were used as sources of *OH and O2*- radicals, respectively. The results show that PDTC effectively scavenges *OH radicals with a reaction rate constant of approximately 2.73 x 10(10) M(-1) s(-1), which is comparable to other efficient *OH radical scavengers, such as ascorbate and glutathione. PDTC is also able to scavenge O2*- radicals. Through its antioxidant properties, PDTC protects against Cr(VI)-induced DNA strand breakage.     

Effects of pirfenidone on the generation of reactive oxygen species in vitro.

Pirfenidone is a newly developed antifibrotic drug that has been reported to retard the progression of pulmonary fibrosis induced by bleomycin and cyclophosphamide in animal models of lung fibrosis. The present in vitro studies using noncellular and cellular systems evaluated the antioxidant and cytotoxic properties of this drug. The Fenton reaction [Fe(II) + H2O2 --> Fe(III) + *OH + OH-] and the xanthine/xanthine oxidase system were used as sources of hydroxyl (*OH) and superoxide anion (O2*-) radicals, respectively. Electron spin resonance spin trapping was used for free radical detection and measurement. The reaction rate of pirfenidone with *OH was found to be 1.63 x 10(10) M(-1) s(-1), which is comparable to several well-established antioxidants, such as ascorbate, glutathione, cysteine, azide, and lipoic acid. Compared to *OH radicals, the O2*- scavenging was less efficient 42.36 M(-1) s(-1) with pirfenidone. Pirfenidone was also effective in inhibiting zymosan-stimulated chemiluminescence. In a noncellular model of lipid peroxidation, pirfenidone inhibited crystalline silica-induced lipid peroxidation. The inhibition of crystalline silica-induced cytotoxic reactions and lipid peroxidation combined with the efficient antioxidant properties of pirfenidone indicate that this agent may express its antifibrotic effects partly through its ability to scavenge reactive oxygen species.     

DNA strand break formation following exposure of bovine pulmonary artery and aortic endothelial cells to reactive oxygen products.

Experiments were performed to investigate the hypothesis that exposure of vascular endothelial cells to low levels of reduced oxygen products results in DNA strand breakage as an early event and to determine if endothelial cells derived from bovine pulmonary artery demonstrate a susceptibility to oxidant injury that is different from that of cells derived from bovine aorta. Endothelial cells grown in culture were exposed to H2O2 (either added directly or generated from glucose oxidase) or superoxide radical (generated from xanthine oxidase), and DNA strand breakage was determined using fluorescent analysis of DNA unwinding. Cell injury was also assessed by measuring the release of lactate dehydrogenase (LDH) or the release of 51Cr from prelabeled cells. Whereas LDH or 51Cr release detected injury resulting from exposure of endothelial cells to greater than or equal to 100 microM H2O2 and was apparent only 2 or more h after exposure, DNA strand breakage was detectable after 15 min of exposure of endothelial cells to 50 microM H2O2. Approximately equivalent DNA strand breakage resulted from exposure to 50 microM H2O2, to 25 mU glucose oxidase, or to 10 mU xanthine oxidase; this injury is similar to that seen following exposure to 10 gray X-radiation. DNA strand breakage following exposure of cells to xanthine oxidase was preventable by catalase but not by superoxide dismutase or hydroxyl radical scavengers, suggesting that H2O2 is the active extracellular oxidant mediating DNA strand breaks. No differences were seen in the susceptibility of pulmonary artery or aortic endothelial cells to oxidant injury.     

Mechanism of neutrophil-induced xanthine dehydrogenase to xanthine oxidase conversion in endothelial cells: evidence of a role for elastase.

Activated neutrophils cause conversion of xanthine dehydrogenase to its oxidase form (xanthine oxidase) in endothelial cells, the mechanism of which may be related to the cytotoxic effect of activated neutrophils. The elastase inhibitors, elastatinal, alpha 1-antitrypsin, and MeO-Suc-(Ala)2-Pro-Val-CH2Cl, significantly inhibited xanthine dehydrogenase to oxidase conversion by phorbol myristate acetate-stimulated neutrophils without inhibition of neutrophil adherence to the endothelial cell monolayer. The role of elastase in this enzyme conversion process was confirmed by the ability of purified elastase to cause conversion of xanthine dehydrogenase to xanthine oxidase in intact endothelial cells (or cell extracts) without causing cytotoxicity. In contrast, cathepsin G failed to cause conversion. The kinetics of conversion induced by elastase was relatively rapid, being essentially completed by 30 min. Upon removal of elastase, the effect was slowly (greater than 12 h) reversible and could be inhibited by cycloheximide treatment. Exposure of endothelial cells to hypoxia failed to enhance the elastase-induced conversion. Treatment of endothelial cells with Ca2+ ionophores failed to cause conversion of xanthine dehydrogenase to oxidase, suggesting that intracellular Ca(2+)-activated proteases are not sufficient to induce this process. Neutrophil-induced xanthine dehydrogenase to oxidase conversion was inhibited by concomitant treatment with antibodies to CD11b. The results suggest that activated neutrophils induce conversion of xanthine dehydrogenase to oxidase by secretion of elastase in close proximity to the endothelial cells and that this intimate contact between the two cell types enables high local concentrations of elastase to be attained, which are sufficient to cause xanthine dehydrogenase to xanthine oxidase conversion.     

On the role of hydroxyl radical and the effect of tetrandrine on nuclear factor--kappaB activation by phorbol 12-myristate 13-acetate

Nuclear factor kappaB (NF-kappaB) is considered to be an important target for therapeutic intervention because of its role in the regulation of proinflammatory and profibrotic mediators. The present study examined the role of hydroxyl (*OH) radical and the effect of tetrandrine, an alkaloid extracted from the Chinese medicinal herb Stephania tetrandra, on NF-kappaB activation by a tumor promoter, phorbol 12-myristate 13-acetate (PMA) in human lymphoid T cells (ie, Jurkat cells). Exogenous superoxide dismutase (SOD) enhanced the NF-kappaB activation by PMA, while catalase blocked it. Formate, a scavenger of *OH radical, also was inhibitory, as was deferoxamine, a metal chelator. These data suggest an important role of *OH radical in PMA-induced NF-kappaB activation. Incubation of the cells with tetrandrine prior to the stimulation of the cells was found to inhibit PMA-induced NF-kappaB activation. Tetrandrine activity was so potent that 50 microM of tetrandrine was sufficient to inhibit activation of NF-kappaB completely. Electron spin resonance (ESR) spin trapping was used to investigate the antioxidant action of tetrandrine using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap. Tetrandrine is an antioxidant for both *OH and superoxide (O2-)radicals. The reaction rate constant of tetrandrine with *OH is 1.4 x 10(10) M(-1)sec(-1), which is comparable with several well established antioxidants, such as ascorbate, glutathione, and cysteine. The Fenton reaction (Fe(II) + H2O2-->Fe(III) + *OH + OH-) and xanthine/xanthine oxidase were used as sources of *OH and O2- radicals. The free radical scavenging activity of tetrandrine is responsible for its inhibition of PMA-induced NF-kappaB activation.     

Respiratory activity of isolated rat liver mitochondria following in vitro exposure to oxygen species: a threshold study

Respiratory activity of isolated rat liver mitochondria was assayed following in vitro exposure to oxygen radicals. Our results show that mitochondrial respiration is more sensitive to O2.(-) than to H2O2. However, ferrous ions drastically enhance the toxicity of the enzymatic system generating H2O2 because of the production of the hydroxyl radicals. A protection against those oxygen species could be given by SOD in the xanthine/xanthine oxidase system and by catalase with the glucose/glucose oxidase system. The most damaging system was the combination of Fe2+ with H2O2. In this case, OH. is formed in a Fenton-like reaction. The fact that the OH. is the most damaging molecule accounts for the finding that catalase and desferrioxamine were efficient protectors in this system. Threshold levels of O2.(-) and H2O2 able to inhibit the mitochondrial respiration have been estimated. It is concluded that under normal respiration such thresholds are not reached in vivo and that the impairment of the mitochondrial respiratory activity does not seem to originate only from the natural free radical production in those organelles. However, if the production of free radicals is such to exceed the defense capability, like under oxidative stress, then the critical threshold can be surpassed and the respiration impaired leading to irreversible damages.     

Comparison between xanthine oxidases from buttermilk and microorganisms regarding their ability to generate reactive oxygen species

Xanthine oxidase (XO) forms uric acid from xanthine. It is assumed that at the same time oxygen is reduced by the XO to reactive oxygen species (ROS), mainly to .O2- and to H2O2. Under certain conditions such ROS can be highly damaging to cellular structures. Therefore, XO was frequently used as a model system, in which the impact of ROS on cellular compounds and structures has been investigated. In this in vitro study xanthine oxidases from buttermilk and from microorganisms were compared regarding their ability to generate ROS. It could be shown that both enzymes are able to transform xanthine to uric acid but differ significantly in their reductive properties to oxygen. XO from buttermilk reduces oxygen to both .O2- and H2O2 whereas XO from microorganisms generates H2O2, but fails to form .O2-. Since .O2- are involved in maintaining transition metal-mediated formation of hydroxyl radicals (.OH) from H2O2, we conclude that XO from microorganisms is therefore largely unsuitable in studies investigating just the interaction of .O2- with other ROS on cellular compounds.     

Ferret tracheal epithelial cells grown in vitro are resistant to lethal injury by activated neutrophils

Airway inflammation is often accompanied by accumulation of polymorphonuclear leukocytes (PMN) as well as epithelial sloughing. To determine whether PMN contribute to epithelial damage in inflammatory states, we examined the interaction of PMN and tracheal epithelial cells in culture. Ferret tracheal epithelial (FTE) cells were grown in primary culture on collagen-coated multiwell dishes. Confluent monolayers were loaded with [51Cr]O4 and exposed to resting and activated neutrophils. There was no significant increase in cell death as assessed by [51Cr]O4 release over 8 h of exposure, at effector (PMN)-to-target cell (epithelial cell) ratios up to 90:1, whether PMN were activated by maximal activating concentrations of phorbol myristate acetate or formylmethionylleucylphenylalanine with or without cytochalasin B. This result was confirmed by using a [3H]leucine release assay as well as by uptake of a supravital dye. However, exposure of FTE cells to activated PMN for 4 h resulted in separation of adjacent cells and formation of gaps in the monolayer, without significant detachment of epithelial cells from the dish. Gap formation was prevented by alpha 1-antitrypsin, N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone, or 10% serum, was mimicked by PMN elastase (24 micrograms/ml), but not by hydrogen peroxide in concentrations up to 10 mM, or superoxide generated by xanthine/xanthine oxidase, and was reversible within 24 h of removal of elastase and exposure to fresh medium. We conclude that activated PMN do not kill FTE cells in culture. However, disruption of the epithelial cell monolayer probably by a proteolytic mechanism can result from exposure to activated PMN and may allow alteration of the epithelial barrier during airway inflammation.     

Walker carcinosarcoma cells damage endothelial cells by the generation of reactive oxygen species.

The passage of circulating tumor cells across vessel walls is an important step in cancer metastasis and is promoted by endothelial injury. Because Walker carcinosarcoma 256 (W256) cells generate oxygen-derived free radicals after cellular activation, the authors tested the hypothesis that these cancer cells can damage endothelial monolayers by producing such reactive oxygen species. To confirm that oxygen-derived radicals can damage endothelial cells, 3H-2-deoxyglucose-labeled human endothelial cell monolayers were exposed to xanthine oxidase in the presence of 0.2 mmol/l xanthine. 3H-2-deoxyglucose release was observed after the addition of xanthine oxidase in concentrations ranging from 6.5 x 10(-3) to 52 x 10(-3) units/ml. The extent of damage correlated with xanthine oxidase-dependent chemiluminescence (r = 0.91). Chemiluminescence assays in the presence of 5 x 10(-5) M luminol confirmed activation of the W256 cells by 1 x 10(-6) M chemotactic peptide fMLP. When fMLP-activated activated W256 cells were incubated with endothelial monolayers, concentrations of 2 x 10(6) to 6 x 10(6) W256 cells/ml were found to cause a 27% increase in the specific release of 2-deoxyglucose after a 90-minute incubation. A small but significant increase in 3H-2-deoxyglucose release also was observed in the absence of fMLP. Detection of 3H-2-deoxyglucose release in the presence of activated or unactivated tumor cells was dependent on preincubating the endothelial cell monolayer with 1 mM buthionine sulfoximine, an inhibitor of glutathione synthesis. Under these conditions, the specific release of 3H-2-deoxyglucose was increased from nondetectable levels to 21%, in the presence of 6.5 x 10(-3) units of the oxidase. Cultured W256 cells promoted isotope release from endothelial cell monolayers when activated with phorbol myristate acetate. Catalase (1000 units/ml) inhibited the tumor cell-induced release of 3H-2-deoxyglucose by 84% whereas superoxide dismutase, even at concentrations of 1 mg/ml, had no effect. A requirement for cell contact was shown because addition of cell-free supernatants from fMLP activated tumor cells did not cause 3H-2-deoxyglucose release and because pretreatment of W256 cells with 1 microM cytochalasin B inhibited their ability to promote isotope release even while increasing tumor cell-generated chemiluminescence threefold. Electron microscopy revealed that fewer cytochalasin B-treated W256 cells were attached to the endothelial cell monolayer than in untreated controls. It is concluded that the W256 tumor cells can damage endothelial cells directly via a mechanism involving production of reactive oxygen species.     

Effects of oxygen metabolites on rat alveolar type II cell viability and surfactant metabolism

Neutrophil-derived reactive oxygen metabolites have been implicated as one mechanism for the cellular injury in the adult respiratory distress syndrome. Previous studies have demonstrated that alveolar lung fluid of patients with adult respiratory distress syndrome has abnormal composition and surface active properties. To examine the effects of oxygen metabolites on the viability and metabolism of type II alveolar pneumocytes, the cellular source of surfactant, isolated rat type II pneumocytes were exposed to reactive oxygen metabolites generated by the enzymatic action of xanthine oxidase upon hypoxanthine. Utilizing a 51Cr release assay to detect cellular death, we found that oxygen metabolites were lethal to type II cells in a dose-dependent manner. To demonstrate that oxygen metabolites were responsible for the toxicity, we assessed the protective effects of catalase and superoxide dismutase, scavengers of hydrogen peroxide and the superoxide anion, respectively. At a xanthine oxidase concentration of 50 mU/ml, catalase reduced the percentage of 51Cr release from 58.9 +/- 3.1% (SEM) to 7.2 +/- 2.3% (p less than 0.0001), whereas superoxide dismutase was without protection (58.9 +/- 3.1% versus 54.2 +/- 1.8% (p greater than 0.05). To determine whether oxygen metabolites also impair surfactant metabolism, we measured the incorporation of [3H]palmitate into the surfactant component disaturated phosphatidylcholine by type II pneumocytes. We found that sublethal amounts of generated oxygen metabolites caused a progressive decrease in the amount of [3H]palmitate incorporated into disaturated phosphatidylcholine. For example, using a xanthine oxidase concentration of 5 mU/ml (which causes no increased 51Cr release), we found that [3H]palmitate incorporation into disaturated phosphatidylcholine fell from a control level of 3.53 +/- 0.22 X 10(5) to 0.66 +/- 0.10 X 10(5) dpm/10(6) cells/4 hours (p less than 0.0001). Both catalase and superoxide dismutase protected the [3H]palmitate incorporation of oxygen metabolite-exposed type II cells. We conclude that reactive oxygen metabolites are injurious to type II pneumocytes and may result in impaired surfactant synthesis even at sublethal doses. Thus, oxygen metabolites generated by stimulated phagocytic cells may be responsible in part for the decreased surfactant that has been observed in adult respiratory distress syndrome.     

Oxidant-mediated damage of Leishmania donovani promastigotes.

Dissemination of Leishmania within the host is related to parasites undergoing unchecked proliferation. We therefore studied the effects of oxidant generating systems on promastigote multiplication by (i) direct determinations of organism proliferation and (ii) the incorporation of [3H]uracil into promastigote nucleoprotein. These two parameters correlated closely as measures of organism replication as demonstrated by parallel suppression of them by the protein synthesis inhibitors puromycin and cycloheximide and the nucleic acid synthesis inhibitors actinomycin D and mitomycin C. Promastigotes showed dose-related susceptibility to reagent and generated hydrogen peroxide (H2O2) as reflected in quantitatively similar decreases in multiplication and [3H]uracil incorporation. These effects were specific for H2O2 as catalase abrogated the dimunition in multiplication. The generation of superoxide anion by acetaldehyde-xanthine oxidase (10 mU/ml) did not alter promastigote replication or nucleoprotein synthesis. These results indicate that Leishmania donovani promastigotes are damaged by H2O2 and that the incorporation of [3H]uracil into promastigote nucleoprotein may be useful for studying the interaction of this parasite with host effector cells.     

Hydrogen peroxide, superoxide, and hydroxyl radicals are involved in the phototoxic action of hematoporphyrin derivative against tumor cells.

This study was aimed to estimate the participation of reactive oxygen species (ROS), other than singlet oxygen (1O2), in the antitumor effect of photodynamic therapy (PDT) with hematoporphyrin derivative (HPD) as well as to determine the ability of photoexcited HPD to the formation of protein peroxides that currently are regarded as a new form of ROS. Studies were performed on Ehrlich ascites carcinoma (EAC) cells, which were loaded with HPD in phosphate-buffered saline and then irradiated with red light at 630 run in the same buffer. Experiments indicated that H2O2 and oxygen radicals could mediate the tumoricidal action of HPD-PDT; we found that photosensitization of EAC cells with HPD leads to the formation of significant amounts of H2O2, superoxide (O2-.), and hydroxyl (OH.) radicals, which along with 1O2 were involved in photoinactivation of the cells in vitro. Our data showed that in EAC cells subjected to HPD-PDT, the generation H2O2, O2-., and OH. could be largely mediated by: (i) an increase in the activity of xanthine oxidase (XOD), due most probably to the conversion of xanthine dehydrogenase (XDH) to XOD via a Ca2+-dependent proteolytic process as well as oxidation of SH groups in XDH; and (ii) photooxidation of some cellular constituents (proteins). Another interesting finding of our studies is that in tumor cells subjected to HPD-PDT the Fenton-like reactions could play an important role in the generation of OH., and that cell-bound Cu/Zn-superoxide dismutase as well as catalase can protect tumor cells against the phototoxic action of HPD. In addition, we clearly demonstrated the ability of photoexcited HPD to the generation of protein peroxides in tumor cells. Studies suggest that 1O2 is the main agent responsible for the generation of protein peroxides in EAC cells treated with HPD-PDT, although other ROS (H2O2, O2-., and OH.) were also implicated in this process. However, further work is needed to clarify the significance of these peroxides in the antitumor effect of PDT with HPD.     

Effects of free radical generation on mouse pial arterioles: probable role of hydroxyl radicals

Mouse pial arterioles were exposed to the free radical-generating reactants acetaldehyde and xanthine oxidase. Concentrations of 0.5 mM acetaldehyde and 0.1 U/ml xanthine oxidase caused reversible dilations, whereas higher concentrations produced initial constrictions followed by reversible dilations. The following free radical scavengers inhibited the dilation when added to the lower concentrations of reactants: superoxide dismutase, a superoxide scavenger; catalase, an H2O2 scavenger; and mannitol, a hydroxyl scavenger. In addition, pretreatment of the animal with dimethyl sulfoxide, a hydroxyl scavenger, also inhibited the response. The scavengers were also tested against either the dilation produced by increased inspired CO2 or against the dilation produced by local application of 10(-3) M papaverine. No significant effect was observed. The data support the hypothesis that hydroxyl radicals can dilate pial arterioles, since all the scavengers can ultimately reduce levels of hydroxyl generated by acetaldehyde plus xanthine oxidase.     

Comparison of smoker and nonsmoker lavage fluid for the rate of association with neutrophil elastase

Cigarette smoking may impair the lung antiprotease screen. To test this hypothesis, the lung lining fluid from 10 smoking and 9 nonsmoking individuals was evaluated for its ability to inhibit neutrophil elastase and porcine pancreatic elastase. To eliminate the possibility of concentration- or purification-induced artifact, unconcentrated bronchoalveolar lavage fluid was used for all experiments. Smokers did not differ significantly from nonsmokers in the antigenic alpha-1-antitrypsin (alpha 1AT) concentrations (0.67 +/- 0.04 versus 0.73 +/- 0.09 nmol alpha 1AT/mg protein), in the neutrophil elastase inhibitory capacity (NEIC) (0.59 +/- 0.03 versus 0.52 +/- 0.05 nmol NEIC/mg protein), or in the porcine pancreatic elastase inhibitory capacity (PPEIC) (0.36 +/- 0.03 versus 0.42 +/- 0.05 nmol PPEIC/mg protein). In contrast, when the PPEIC/NEIC ratio was evaluated, smoker lung lining fluid exhibited a relative defect (0.64 +/- 0.06 smokers versus 0.80 +/- 0.05 nonsmokers, P less than 0.05). In agreement with the smokers' defect in the PPEIC/NEIC ratio, the kinetics of association (Ka) of lung lining fluid for neutrophil elastase was 0.38 +/- 0.3 x 10(7) M-1 s-1 from smokers and 0.58 +/- 0.05 x 10(7) M-1 s-1 from nonsmokers (P less than 0.002). Thus for a given amount of neutrophil elastase, smoker lung lining fluid took approximately 1.5 times longer to inhibit neutrophil elastase. These findings suggest that cigarette smoking is associated with a subtle defect in the antiprotease screen of the lower respiratory tract, recognizable by time-dependent measures of anti-neutrophil elastase function.     

Serum xanthine oxidase: origin, regulation, and contribution to control of trypanosome parasitemia

African trypanosomiasis is caused by Salivarian trypanosomes, tsetse fly-transmitted protozoa that inhabit the blood plasma, lymph and interstitial fluids, and, in the case of Trypanosoma brucei species, also the cerebrospinal fluid of mammal hosts. Trypanosomiasis in people and domestic animals manifests as recurring waves of parasites in the blood and is typically fatal. In contrast, trypanosomiasis in Cape buffaloes, which are naturally selected to resist the disease, is characterized by the development of only one or a few waves of parasitemia, after which the infection becomes cryptic, being maintained by the presence of 1-20 mammal-infective organisms/ml of blood. The control of the acute phase of parasitemia in Cape buffaloes correlates with a decline in blood catalase activity and the generation of trypanocidal H(2)O(2) in serum during the catabolism of endogenous purine by xanthine oxidase. Here we review features of this response, and of trypanosome metabolism, that facilitate H(2)O(2)-mediated killing of the parasites with minimal damage to the host. We also discuss the origin and regulation of serum xanthine oxidase and catalase, and show how recovery of serum catalase in infected Cape buffaloes precludes a role for H(2)O(2) in the long-term, stable suppression of trypanosome parasitemia.     

Response of Leishmania chagasi promastigotes to oxidant stress.

At the onset of infection, Leishmania promastigotes are phagocytized by mammalian macrophages. They must survive despite exposure to toxic oxidants such as hydrogen peroxide (H2O2) and superoxide (.O2-) generated during phagocytosis. We investigated the effects of these oxidants on Leishmania chagasi promastigotes and promastigote mechanisms for oxidant resistance. According to spin trapping and electron paramagnetic resonance spectrometry, .O2- could be generated by exposure of promastigotes to the redox-cycling compound menadione. Incubation in either menadione or H2O2 caused a concentration-dependent loss of promastigote viability. However, incubation in sublethal concentrations of H2O2 or menadione caused a stress response in promastigotes. This oxidant-induced response was associated with an increase in the amount of heat shock protein hsp70. Induction of a stress response by exposure of promastigotes either to heat shock or to sublethal oxidants (H2O2 or menadione) caused promastigotes to become more resistant to H2O2 toxicity. Sublethal menadione also caused promastigotes to become more virulent in a BALB/c mouse model of leishmaniasis. We previously correlated H2O2 cytotoxicity for promastigotes with the formation of hydroxyl radical (.OH) from H2O2. However, according to electron paramagnetic resonance spectrometry, the increase in H2O2 resistance after exposure to sublethal oxidants was not associated with diminished generation (i.e., scavenging) of .OH. These data suggest that there is a cross-protective stress response that occurs after exposure of L. chagasi promastigotes to heat shock or to sublethal H2O2 or .O2-, exposures that also occur during natural infection. This response results in increased resistance to H2O2 toxicity and increased virulence for a mammalian host.     

Generation of hydrogen peroxide by Candida albicans and influence on murine polymorphonuclear leukocyte activity.

Iodide fixation by murine polymorphonuclear leukocytes (PMN) incubated with viable Candida albicans blastoconidia increases directly with yeast cell concentration up to about 3 x 10(6) cells per ml, but above this concentration bound activity declines dramatically. To understand the basis for this decline, we examined the oxidative metabolism of fungi and stimulated PMN and found some remarkable similarities between these cell types. Both produced 14CO2 when incubated with [1-14C]glucose, both reduced cytochrome c, and both fixed radiolabeled iodide, although the fungi required exogenous lactoperoxidase. In dose-response experiments, iodination by fungi with lactoperoxidase was identical to that with PMN, i.e., the maximum bound activity occurred in cultures with 10(6) to 3 x 10(6) blastoconidia per ml. Iodination by fungi with lactoperoxidase was reduced when blastoconidia were incubated at 25 degrees C or in the presence of catalase and the metabolic inhibitors rotenone, antimycin A, and 2-deoxyglucose. Results from assays for oxidation of scopoletin and o-dianisidine showed that 10(6) blastoconidia in 1.0 ml of medium released 0.5 to 0.7 nmol of H2O2 after 1 h, but 3 X 10(6) and 10(7) cells released significantly less H2O2. These results suggest that iodide fixation by PMN and low numbers of fungal cells may reflect a cooperative effort, with fungi generating some H2O2 that reacts with the myeloperoxidase released from the PMN. With high concentrations of blastoconidia, H2O2 activity appeared to be specifically inhibited, possibly to protect fungal cells from damage.