Bacteria form intracellular free radicals in response to paraquat and streptonigrin. Demonstration of the potency of hydroxyl radical

The generation of oxygen reduction products by Neisseria gonorrhoeae FA1090 upon exposure to streptonigrin (SNG) and paraquat (PQ2+) and their toxicity was examined. N. gonorrhoeae exhibited maximal cyanide-insensitive respiration, which was employed as an indicator of superoxide (O2-) formation, in the presence of 0.064 mM streptonigrin and 90 mM PQ2+, respectively. Using the concentrations of SNG and PQ2+ described above, complete lethality (greater than 10(8) cells/ml) was observed among cells exposed to SNG, whereas PQ2+ reduced viability by only 3 logs. In an attempt to determine the oxygen radical species generated by gonococci when exposed to SNG, dimethyl sulfoxide, Fe3+, KCN, and the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), we were able to detect .OH manifested as the methyl adduct (DMPO-CH3). The production of the latter species was not inhibited by catalase, suggesting intracellular .OH generation. When PQ2+ was substituted for SNG, only low levels of DMPO-CH3 were observed, the production of which ceased within 8 min. SNG and PQ2+, added to a O2(-)- generating system in the presence of Fe3+, promoted increased .OH generation. The iron chelator diethyl-enetriaminepentaacetic acid enhanced the generation of spin-trapped .OH and O2- in the presence of PQ2+. The addition of catalase to this system, however, eliminated the DMPO-CH3 signal, showing that the .OH in this system was extracellular. PQ2+-mediated generation of extracellular .OH in the presence of Fe3+-diethylenetriaminepentaacetic acid EDTA did not enhance the killing of gonococci by PQ2+. These data show that the lethality of SNG relative to PQ2+ is due to the inherent ability of SNG to catalyze the formation of critical levels of intracellular .OH, detectable through the use of spin trapping techniques.     

Trichoderma spp. and Bacillus spp. as growth promoters in maize (Zea mays L.)

Microbes that are beneficial to plants are used to enhance the crop growth, yield and are alternatives to chemical fertilizers. Trichoderma and Bacillus are the predominant plant growth-promoting fungi and bacteria. The objective of this study was select, characterize, and evaluate isolates of Trichoderma spp. and Bacillus spp. native from the northern region of Sinaloa, Mexico, and assess their effect on growth promotion in maize (Zea mays L.). In greenhouse conditions, four Trichoderma isolates and twenty Bacillus isolates, as well as two controls, were tested in a completely randomized design with three replicates. We selected the two best strains of Trichoderma and Bacillus: TB = Trichoderma asperellum, TF = Trichoderma virens, B14 = Bacillus cereus sensu lato and B17 = Bacillus cereus, which were evaluated in the field in a completely randomized blocks in factorial arrangement design with three replicates applying different rates of nitrogen fertilizer (0, 150 kg N/ha, and 300 kg N/ha). Treatments 5 (B17 = B. cereus) and 11 (TF = T. virens) both fertilized with 150 kg N/ha showed similar yields and they did not reveal significant differences from the treatments fertilized with 300 kg N/ha. This indicated that treatment 5 (B17= B. cereus with 150 kg N/ha) and treatment 11 (TF= T. virens with 150 kg N/ha) were efficient as growth promoters, by not showing significant differences in root volume and dry weight of foliage. The results indicated a reduction of 50% in the rate of nitrogen to fertilizer required for maize (Zea mays L.) crops. These microorganisms Trichoderma and Bacillus could be an alternative to reduce the use of chemical fertilizers in maize.     

Doxorubicin inhibits oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) by a lactoperoxidase/H(2)O(2) system by reacting with ABTS-derived radical

The effect of doxorubicin on oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) by lactoperoxidase and hydrogen peroxide has been investigated. It was found that: (1) oxidation of ABTS to its radical cation (ABTS*(+)) is inhibited by doxorubicin as evidenced by its induction of a lag period, duration of which depends on doxorubicin concentration; (2) the inhibition is due to doxorubicin hydroquinone reducing the ABTS*(+) radical (stoichiometry 1: 1.8); (3) concomitant with the ABTS*(+) reduction is oxidation of doxorubicin; only when the doxorubicin concentration decreases to a near zero level, net oxidation of ABTS could be detected; (4) oxidation of doxorubicin leads to its degradation to 3-methoxysalicylic acid and 3-methoxyphthalic acid; (5) the efficacy of doxorubicin to quench ABTS*(+) is similar to the efficacy of p-hydroquinone, glutathione and Trolox C. These observations support the assertion that under certain conditions doxorubicin can function as an antioxidant. They also suggest that interaction of doxorubicin with oxidants may lead to its oxidative degradation.     

Do humans neutrophils form hydroxyl radical? Evaluation of an unresolved controversy

Hydroxyl radical is a potent oxidizing agent of potential importance in human pathobiology. Since neutrophilic phagocytes make superoxide and hydrogen peroxide during phagocytosis, it has been proposed that hydroxyl radical is also formed. In this paper we review the literature which supports or refutes formation of hydroxyl radical by neutrophils and the mechanism(s) by which this radical might be formed. We conclude that there is no definitive proof for hydroxyl radical formation by neutrophils. In fact, neutrophil release of lactoferrin and myeloperoxidase appears to limit formation of this radical. Future studies are likely to determine whether superoxide released by neutrophils interacts with target substrates to allow formation of hydroxyl radical.     

Inactivation of the potent Pseudomonas aeruginosa cytotoxin pyocyanin by airway peroxidases and nitrite

Pyocyanin (1-hydroxy-N-methylphenazine, PCN) is a cytotoxic pigment and virulence factor secreted by the human bacterial pathogen, Pseudomonas aeruginosa. Here, we report that exposure of PCN to airway peroxidases, hydrogen peroxide (H(2)O(2)), and NaNO(2) generates unique mononitrated PCN metabolites (N-PCN) as revealed by HPLC/mass spectrometry analyses. N-PCN, in contrast to PCN, was devoid of antibiotic activity and failed to kill Escherichia coli and Staphylococcus aureus. Furthermore, in contrast to PCN, intratracheal instillation of N-PCN into murine lungs failed to induce a significant inflammatory response. Surprisingly, at a pH of ～7, N-PCN was more reactive than PCN with respect to NADH oxidation but resulted in a similar magnitude of superoxide production as detected by electron paramagnetic resonance and spin trapping experiments. When incubated with Escherichia coli or lung A549 cells, PCN and N-PCN both led to superoxide formation, but lesser amounts were detected with N-PCN. Our results demonstrate that PCN that has been nitrated by peroxidase/H(2)O(2)/NO(2)(-) systems possesses less cytotoxic/proinflammatory activity than native PCN. Yield of N-PCN was decreased by the presence of the competing physiological peroxidase substrates (thiocyonate) SCN(-) (myeloperoxidase, MPO, and lactoperoxidase, LPO) and Cl(-) (MPO), which with Cl(-) yielded chlorinated PCNs. These reaction products also showed decreased proinflammatory ability when instilled into the lungs of mice. These observations add important insights into the complexity of the pathogenesis of lung injury associated with Pseudomonas aeruginosa infections and provide additional rationale for exploring the efficacy of NO(2)(-) in the therapy of chronic Pseudomonas aeruginosa airway infection in cystic fibrosis.     

The spin trapping of superoxide with M4PO (3,3,5,5-tetramethylpyrroline-N-oxide)

The spin trap 3,3,5,5-tetramethylpyrroline-N-oxide (M4PO) reacts with superoxide to produce a short-lived spin adduct (M4PO/.OOH, AN = 14.0 G, AH = 6.5 G) that decays by a first-order process (t1/2 approximately 35 s at pH 7.4). This short lifetime may limit its usefulness in studies of superoxide.     

Spin-trapping and human neutrophils. Limits of detection of hydroxyl radical

Using the spin trap, 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and an excess of dimethyl sulfoxide, we previously reported that in the absence of an exogenous iron catalyst, human neutrophils will not generate hydroxyl radical, manifested as the catalse-inhibitable methyl radical spin-trapped adduct, 2,2,5-trimethyl-1-pyrrolidinyloxy (DMPO-CH3) (Britigan, B. E., Rosen, G. M., Chai, Y., and Cohen, M. S. (1986) J. Biol. Chem. 261, 4426-4431). However, superoxide destroys the preformed hydroxyl radical spin-trapped adduct, 2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxy (DMPO-OH), and DMPO-CH3. The present study was undertaken to better resolve the limits of sensitivity of the spin-trapping method. Photolytically generated DMPO-CH3 and DMPO-OH slowly decomposed in the presence of a low flux (1 microM/min) of enzymatically (xanthine/xanthine oxidase)-generated superoxide, but more rapid decomposition of these adducts occurred with higher superoxide flux (5 microM/min). Inclusion of cysteine markedly increased the rate of DMPO-OH and DMPO-CH3 decomposition, masking the effect of superoxide alone. The addition of varying concentrations of superoxide dismutase did not lead to increased formation of DMPO-OH or DMPO-CH3, as should have occurred if these adducts were being destroyed by superoxide. As a positive control, we employed an iron-supplemented system with phorbol 12-myristate 13-acetate-stimulated neutrophils or xanthine/xanthine oxidase to generate DMPO-CH3. Addition of superoxide dismutase increased the magnitude of DMPO-CH3, primarily by increasing the rate of hydrogen peroxide formation, and to a lesser extent by prolonging the half-life of DMPO-CH3. Although spin-trapped adducts can be destroyed by a high concentration of superoxide, or by lower concentrations of superoxide in the presence of thiol-containing compounds, our results demonstrate that such decomposition does not interfere with the ability of the spin-trapping method to detect hydroxyl radical generated by human neutrophils. These data do not support the capacity of neutrophils to generate hydroxyl radical in the absence of an exogenous Haber-Weiss catalyst.     

Hydrogen peroxide-induced apoptosis of HL-60 human leukemia cells is mediated by the oxidants hypochlorous acid and chloramines

We set out to identify whether HOCl, which is generated from H(2)O(2) /MPO/Cl(-), is a proximal mediator of H(2)O(2) programmed cell death in the HL-60 human leukemia cell. We found that authentic HOCl induces apoptosis in the HL-60 cell. Both the addition of methionine, an HOCl scavenger, and the removal of Cl(-) from the medium to prevent the formation of HOCl inhibited H(2)O(2)-induced apoptosis. HL-60 cells underwent apoptosis when exposed to HOCl in full medium, which gives rise to chloramines by the reaction of HOCl with amine groups, but not by HOCl in the amine-free HBSS, in which HOCl but not chloramines can be detected. Authentic chloramines induced apoptosis in this cell line in a concentration-dependent manner and at concentrations lower than HOCl. Full medium exposed to HOCl for 24 h would support methionine noninhibitable apoptosis, but did not react with 2-nitro-5-thiobenzoic acid (TNB), raising the possibility that the final inducer is a nonoxidant formed from HOCl and chloramines. We conclude that the signal for apoptosis induced by H(2)O(2) in the MPO-containing HL-60 cell involves the reaction of the diffusible oxidant HOCl with amines producing chloramines and a subsequent non-TNB-reactive product.     

Intraphagosomal Mycobacterium tuberculosis acquires iron from both extracellular transferrin and intracellular iron pools. Impact of interferon-gamma and hemochromatosis

Mycobacterium tuberculosis multiplies within the macrophage phagosome and requires iron for growth. We examined the route(s) by which intracellular M. tuberculosis acquires iron. During intracellular growth of the virulent Erdman M. tuberculosis strain in human monocyte-derived macrophages (MDM), M. tuberculosis acquisition of (59)Fe from transferrin (TF) provided extracellularly (exogenous source) was compared with acquisition when MDM were loaded with (59)Fe from TF prior to M. tuberculosis infection (endogenous sources). M. tuberculosis (59)Fe acquisition required viable bacteria and was similar from exogenous and endogenous sources at 24 h and greater from exogenous iron at 48 h. Interferon-gamma treatment of MDM reduced (59)Fe uptake from TF 51% and TF receptor expression by 34%. Despite this, intraphagosomal M. tuberculosis iron acquisition in IFN-gamma-treated cells was decreased by only 30%. Macrophages from hereditary hemochromatosis patients have altered iron metabolism. Intracellular M. tuberculosis acquired markedly less iron in MDM from these individuals than in MDM from healthy donors, regardless of the iron source (exogenous and endogenous): 36 +/- 3.8% and 17 +/- 9.6% of control, respectively. Thus, intraphagosomal M. tuberculosis can acquire iron from both extracellular TF and endogenous macrophage sources. Acquisition of iron from macrophage cytoplasmic iron pools may be critical for the intracellular growth of M. tuberculosis. This acquisition is altered by IFN-gamma treatment to a small extent, but is markedly reduced in macrophages from hemochromatosis patients.     

Phenazine-1-carboxylic acid,a secondary metabolite of Pseudomonas aeruginosa,alters expression of immunomodulatory proteins by human airway epithelial cells

Pseudomonas aeruginosa is a gram-negative bacterium that causes both acute and chronic lung disease in susceptible patient populations. P. aeruginosa secretes numerous proteins and secondary metabolites, many of which have biological effects that likely contribute to disease pathogenesis. An unidentified small-molecular-weight factor was previously reported to increase IL-8 release both in vitro and in vivo. To identify this factor, we subjected the <3-kDa fraction from P. aeruginosa-conditioned medium to HPLC analysis. A peak fraction that stimulated IL-8 release was found by mass spectrometry to have a molecular mass (MM) of 224 Da. On the basis of this MM and other biochemical properties, we hypothesized that the factor was phenazine-1-carboxylic acid (PCA). Subsequent studies and comparison with purified PCA confirmed this hypothesis. Purified PCA exhibited a number of biological effects in human airway epithelial cells, including increasing IL-8 release and ICAM-1 expression, as well as decreasing RANTES and monocyte chemoattractant protein-1 (MCP-1) release. PCA also increased intracellular oxidant formation as measured by electron paramagnetic resonance and by an intracellular oxidant-sensitive probe. Antioxidants inhibited PCA-dependent increases in IL-8 and ICAM-1, suggesting that oxidants contributed to these effects. However, in contrast to the related phenazine compound pyocyanin, PCA did not oxidize NAD(P)H at physiologically relevant pH, providing preliminary evidence that PCA and pyocyanin may have distinct redox chemistries within the cell. Thus PCA is a biologically active factor secreted by P. aeruginosa that has several activities that could alter the host immune and inflammatory response and thereby contribute to bacterial disease pathogenesis.