Ronald P. Mason

Pseudomonas aeruginosa Anaerobic Respiration in Biofilms: Relationships to Cystic Fibrosis Pathogenesis

Recent data indicate that cystic fibrosis (CF) airway mucus is anaerobic. This suggests that Pseudomonas aeruginosa infection in CF reflects biofilm formation and persistence in an anaerobic environment. P. aeruginosa formed robust anaerobic biofilms, the viability of which requires rhl quorum sensing and nitric oxide (NO) reductase to modulate or prevent accumulation of toxic NO, a byproduct of anaerobic respiration. Proteomic analyses identified an outer membrane protein, OprF, that was upregulated approximately 40-fold under anaerobic versus aerobic conditions. Further, OprF exists in CF mucus, and CF patients raise antisera to OprF. An oprF mutant formed poor anaerobic biofilms, due, in part, to defects in anaerobic respiration. Thus, future investigations of CF pathogenesis and therapy should include a better understanding of anaerobic metabolism and biofilm development by P. aeruginosa.

The role of Kupffer cell oxidant production in early ethanol-induced liver disease.

Considerable evidence for a role of Kupffer cells in alcoholic liver disease has accumulated and they have recently been shown to be a predominant source of free radicals. Several approaches including pharmacological agents, knockout mice, and viral gene transfer have been used to fill critical gaps in understanding key mechanisms by which Kupffer cell activation, oxidant formation, and cytokine production lead to liver damage and subsequent pathogenesis. This review highlights new data in support of the hypothesis that Kupffer cells play a pivotal role in hepatotoxicity due to ethanol by producing oxidants via NADPH oxidase.

Phenoxyl Free Radical Formation during the Oxidation of the Fluorescent Dye 2′,7′-Dichlorofluorescein by Horseradish Peroxidase POSSIBLE CONSEQUENCES FOR OXIDATIVE STRESS MEASUREMENTS

The oxidation of the fluorescent dye 2′,7′-dichlorofluorescein (DCF) by horseradish peroxidase was investigated by optical absorption, electron spin resonance (ESR), and oxygen consumption measurements. Spectrophotometric measurements showed that DCF could be oxidized either by horseradish peroxidase-compound I or -compound II with the obligate generation of the DCF phenoxyl radical (DCF⋅). This one-electron oxidation was confirmed by ESR spin-trapping experiments. DCF⋅ oxidizes GSH, generating the glutathione thiyl radical (GS⋅), which was detected by the ESR spin-trapping technique. In this case, oxygen was consumed by a sequence of reactions initiated by the GS⋅ radical. Similarly, DCF⋅ oxidized NADH, generating the NAD⋅ radical that reduced oxygen to superoxide (O⨪2), which was also detected by the ESR spin-trapping technique. Superoxide dismutated to generate H2O2, which reacted with horseradish peroxidase, setting up an enzymatic chain reaction leading to H2O2 production and oxygen consumption. In contrast, when ascorbic acid reduced the DCF phenoxyl radical back to its parent molecule, it formed the unreactive ascorbate anion radical. Clearly, DCF catalytically stimulates the formation of reactive oxygen species in a manner that is dependent on and affected by various biochemical reducing agents. This study, together with our earlier studies, demonstrates that DCFH cannot be used conclusively to measure superoxide or hydrogen peroxide formation in cells undergoing oxidative stress.

Protein Oxidation of Cytochrome c by Reactive Halogen Species Enhances Its Peroxidase Activity

Reactive halogen species (RHS; X2 and HOX, where X represents Cl, Br, or I) are metabolites mediated by neutrophil activation and its accompanying respiratory burst. We have investigated the interaction between RHS and mitochondrial cytochrome c (cyt c) by using electrospray mass spectrometry and electron spin resonance (ESR). When the purified cyt c was reacted with an excess amount of hypochlorous acid (HOCl) at pH 7.4, the peroxidase activity of cytc was increased by 4.5-, 6.9-, and 8.6-fold at molar ratios (HOCl/cyt c) of 2, 4, and 8, respectively. In comparison with native cyt c, the mass spectra obtained from the HOCl-treated cyt c revealed that oxygen is covalently incorporated into the protein as indicated by molecular ions of m/z = 12,360 (cyt c), 12,376 (cyt c + O), and 12,392 (cyt c + 2O). Using tandem mass spectrometry, a peptide (obtained from the tryptic digests of HOCl-treated cyt c) corresponding to the amino acid sequence MIFAGIK, which contains the methionine that binds to the heme, was identified to be involved in the oxygen incorporation. The location of the oxygen incorporation was unequivocally determined to be the methionine residue, suggesting that the oxidation of heme ligand (Met-80) by HOCl results in the enhancement of peroxidase activity of cyt c. ESR spectroscopy of HOCl-oxidized cyt c, when reacted with H2O2 in the presence of the nitroso spin trap 2-methyl-2-nitrosopropane (MNP), yielded more immobilized MNP/tyrosyl adduct than native cytc. In the presence of H2O2, the peroxidase activity of HOCl-oxidized cyt c exhibited an increasing ability to oxidize tyrosine to tyrosyl radical as measured directly by fast flow ESR. Titration of both native cyt cand HOCl-oxidized cyt c with various amounts of H2O2 indicated that the latter has a decreased apparent K m for H2O2, implicating that protein oxidation of cyt c increases its accessibility to H2O2. HOCl-oxidized cytc also displayed an impaired ability to support oxygen consumption by the purified mitochondrial cytochrome coxidase, suggesting that protein oxidation of cyt c may break the electron transport chain and inhibit energy transduction in mitochondria.

Peroxidation of a Specific Tryptophan of Metmyoglobin by Hydrogen Peroxide

Globin-centered radicals at tyrosine and tryptophan residues and a peroxyl radical at an unknown location have been reported previously as products of the reaction of metmyoglobin with hydrogen peroxide. The peroxyl radical is shown here to be localized on tryptophan through the use of recombinant sperm whale myoglobin labeled with 13C at the indole ring C-3. Peroxyl radical formation was not prevented by site-directed mutations that replaced all three tyrosines, the distal histidine, or tryptophan 7 with non-oxidizable residues. In contrast, mutation of tryptophan 14 prevents peroxyl radical formation, implicating tryptophan 14 as the specific site of the peroxidation.

Acute cadmium exposure induces stress-related gene expression in wild-type and metallothionein-I/II-null mice.

This study examined the effect of acute cadmium on stress-related gene expression and free radical production in wild-type and metallothionein-I/II-null (MT-null) mice. Atlas Toxicology arrays showed that acute cadmium (40 micromol/kg as CdCl(2), ip for 3 h) markedly increased the expression of genes encoding heat-shock proteins, heme oxygenase-1, and genes in response to DNA damage/repair. The expression of genes encoding cytochrome P450 enzymes, UDP-glucuronosyltransferases, Mn-superoxide dismutase, and catalase was suppressed by cadmium. MT-null mice were more sensitive than wild-type mice to cadmium-induced, stress-related gene expression, in accord with greater activation of transcription factor AP-1 and phosphorylated JNK and ERK. To evaluate free radical production, mice were simultaneously given the spin trap agent, N-tert-butyl-alpha-phenylnitrone (PBN, 250 mg in DMSO/kg, ip) with cadmium, and livers were removed 30 min later for PBN-trapped radical extraction with chloroform:methanol (2:1), and detected with electron spin resonance (ESR). Cadmium treatment caused detectable ESR signals for PBN adducts as well as lipid peroxidation in the liver similarly in both wild-type and MT-null mice. Thus, the mechanism of acute cadmium toxicity involves multiple facets including oxidative damage and aberrant gene expression, and absence of MT exacerbates Cd-induced aberrant gene expression.

The Fate of the Oxidizing Tyrosyl Radical in the Presence of Glutathione and Ascorbate IMPLICATIONS FOR THE RADICAL SINK HYPOTHESIS

Cellular systems contain as much as millimolar concentrations of both ascorbate and GSH, although the GSH concentration is often 10-fold that of ascorbate. It has been proposed that GSH and superoxide dismutase (SOD) act in a concerted effort to eliminate biologically generated radicals. The tyrosyl radical (Tyr⋅) generated by horseradish peroxidase in the presence of hydrogen peroxide can react with GSH to form the glutathione thiyl radical (GS⋅). GS⋅ can react with the glutathione anion (GS−) to form the disulfide radical anion (GSSG⨪). This highly reactive disulfide radical anion will reduce molecular oxygen, forming superoxide and glutathione disulfide (GSSG). In a concerted effort, SOD will catalyze the dismutation of superoxide, resulting in the elimination of the radical. The physiological relevance of this GSH/SOD concerted effort is questionable. In a tyrosyl radical-generating system containing ascorbate (100 μm) and GSH (8 mm), the ascorbate nearly eliminated oxygen consumption and diminished GS⋅ formation. In the presence of ascorbate, the tyrosyl radical will oxidize ascorbate to form the ascorbate radical. When measuring the ascorbate radical directly using fast-flow electron spin resonance, only minor changes in the ascorbate radical electron spin resonance signal intensity occurred in the presence of GSH. These results indicate that in the presence of physiological concentrations of ascorbate and GSH, GSH is not involved in the detoxification pathway of oxidizing free radicals formed by peroxidases.

Nitroxide Radical Adducts in Biology: Chemistry, Applications, and Pitfalls

Spin trapping is a technique in which a reactive free radical reacts with a double bond of a diamagnetic compound, the spin trap, to form a less reactive free radical, the spin adduct. Spin trapping is often necessary when the primary free radical cannot be observed by conventional ESR either because of low concentration or very short radical relaxation times which lead to very broad lines (Janzen, 1980).

Nitric Oxide-forming Reaction between the Iron-N-Methyl-d-glucamine Dithiocarbamate Complex and Nitrite

The objective of this study was to elucidate the origin of the nitric oxide-forming reactions from nitrite in the presence of the iron-N-methyl-d-glucamine dithiocarbamate complex ((MGD)2Fe2+). The (MGD)2Fe2+ complex is commonly used in electron paramagnetic resonance (EPR) spectroscopic detection of NO bothin vivo and in vitro. Although it is widely believed that only NO can react with (MGD)2Fe2+ complex to form the (MGD)2Fe2+·NO complex, a recent article reported that the (MGD)2Fe2+ complex can react not only with NO, but also with nitrite to produce the characteristic triplet EPR signal of (MGD)2Fe2+·NO (Hiramoto, K., Tomiyama, S., and Kikugawa, K. (1997) Free Radical Res. 27, 505–509). However, no detailed reaction mechanisms were given. Alternatively, nitrite is considered to be a spontaneous NO donor, especially at acidic pH values (Samouilov, A., Kuppusamy, P., and Zweier, J. L. (1998) Arch Biochem. Biophys. 357, 1–7). However, its production of nitric oxide at physiological pH is unclear. In this report, we demonstrate that the (MGD)2Fe2+ complex and nitrite reacted to form NO as follows: 1) (MGD)2Fe2·NO complex was produced at pH 7.4; 2) concomitantly, the (MGD)3Fe3+ complex, which is the oxidized form of (MGD)2Fe2+, was formed; 3) the rate of formation of the (MGD)2Fe2+·NO complex was a function of the concentration of [Fe2+]2, [MGD], [H+] and [nitrite].

An electron spin resonance spin-trapping investigation of the free radicals formed by the reaction of mitochondrial cytochrome c oxidase with H2O2.

The reaction of purified bovine mitochondrial cytochrome c oxidase (CcO) and hydrogen peroxide was studied using the ESR spin-trapping technique. A protein-centered radical adduct was trapped by 5,5-dimethyl-1-pyrrolineN-oxide and was assigned to a thiyl radical adduct based on its hyperfine coupling constants of a N = 14.7 G and a β H = 15.7 G. The ESR spectra obtained using the nitroso spin traps 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS) and 2-methyl-2-nitrosopropane (MNP) indicated that both DBNBS/⋅CcO and MNP/⋅CcO radical adducts are immobilized nitroxides formed by the trapping of protein-derived radicals. Alkylation of the free thiols on the enzyme with N-ethylmaleimide (NEM) prevented 5,5-dimethyl-1-pyrroline N-oxide adduct formation and changed the spectra of the MNP and DBNBS radical adducts. Nonspecific protease treatment of MNP-d 9/⋅NEM-CcO converted its spectrum from that of an immobilized nitroxide to an isotropic three-line spectrum characteristic of rapid molecular motion. Super-hyperfine couplings were detected in this spectrum and assigned to the MNP/⋅tyrosyl adduct(s). The inhibition of either CcO or NEM-CcO with potassium cyanide prevented detectable MNP adduct formation, indicating heme involvement in the reaction. The results indicate that one or more cysteine residues are the preferred reductant of the presumed ferryl porphyrin cation radical residue intermediate. When the cysteine residues are blocked with NEM, one or more tyrosine residues become the preferred reductant, forming the tyrosyl radical.