Nebulized thiocyanate improves lung infection outcomes in mice.

BACKGROUND AND PURPOSE: Nebulized saline solutions are used in the treatment of multiple pulmonary diseases including cystic fibrosis (CF), asthma and chronic obstructive pulmonary disease (COPD). The benefits of these therapies include improved lung function, phlegm clearance and fewer lung infections. The thiocyanate anion (SCN) is a normal component of the airway epithelial lining fluid (ELF) secreted by pulmonary epithelia with antioxidant and host defence functions. We sought to test if SCN could be nebulized to combat lung infection by bolstering innate immune defence and antioxidant capacity. EXPERIMENTAL APPROACH: We established an effective antioxidant concentration of SCN in vitro using a bronchiolar epithelial cell line. We then developed a nebulization method of SCN in mice that increased ELF SCN above this concentration up to 12 h and used this method in a prolonged Pseudomonas aeruginosa infection model to test if increasing SCN improved host defence and infection outcomes. KEY RESULTS: SCN protected against cytotoxicity in vitro from acute and sustained exposure to inflammation-associated oxidative stress. Nebulized SCN effectively reduced bacterial load, infection-mediated morbidity and airway inflammation in mice infected with P. aeruginosa. SCN also sustained adaptive increases in reduced GSH in infected mice. CONCLUSIONS AND IMPLICATIONS: SCN is a dually protective molecule able to both enhance host defence and decrease tissue injury and inflammation as an antioxidant. Nebulized SCN could be developed to combat lung infections and inflammatory lung disease.

Novel antioxidants zolimid and AEOL11201 ameliorate colitis in rats.

The mechanism of tissue damage in ulcerative colitis (UC) is unknown. However, recent evidence suggests that reactive oxygen species (ROS) are critical mediators of inflammation, and tissue damage in UC and antioxidants could be beneficial in the treatment of UC. Our aim was to evaluate the effects of two new antioxidants, Zolimid and AEOL11201 on experimental colitis. Antioxidants or vehicle were given to rats for five days after induction of colitis by intrarectal administration of 4% acetic acid. Severity of colitis was assessed on day 5. Zolimid and AEOL11201 significantly improved acetic acid-induced colitis. Both Zolimid and AEOL11201 significantly decreased the severity of diarrhea, and severity of macroscopic and histological changes in the colon. Both agents also significantly decreased colonic MPO levels. In conclusion, Zolimid and AEOL11201 are effective antiinflammatory agents in an animal model of colitis. Further studies are needed to evaluate their beneficial therapeutic effects in patients with UC.

2-methoxyestradiol does not inhibit superoxide dismutase.

It has been reported in the literature that the endogenous estrogen metabolite 2-methoxyestradiol (2-ME) inhibits both manganese and copper,zinc superoxide dismutases (Mn and Cu,Zn SODs) and that this mechanism is responsible for 2-ME's ability to kill cancer cells. In fact, as demonstrated using several SOD assays including pulse radiolysis, 2-ME does not inhibit SOD but rather interferes with the SOD assay originally used. Nevertheless, as confirmed by aconitase inactivation measurements and lactate dehydrogenase release in human leukemia HL-60 cells, 2-ME does increase superoxide production in these cells and is more toxic than its non-O-methylated precursor 2-hydroxyestradiol. Other mechanisms previously suggested in the literature may explain 2-ME's ability to increase intracellular superoxide levels in tumor cells.

Attenuation of bleomycin-induced pulmonary fibrosis by a catalytic antioxidant metalloporphyrin.

Oxidative stress plays an important role in the development of fibrotic responses in the lung. However, it is not clear whether inhibiting oxidative stress with antioxidants can attenuate fibrotic processes in the lung. The objective of these studies was to test whether the catalytic antioxidant porphyrin manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) could protect mice against bleomycin-induced lung fibrosis. A 10 mg/kg intraperitoneal dose of MnTBAP was established as safe and had a serum and lung half-life of 9.5 h in mice. Based on this data, four groups of mice were given one dose of bleomycin (3.2 U/kg, intratracheal) or saline and MnTBAP (5 mg/kg, intraperitoneal) or saline twice daily for 14 d. Lung fibrosis was assessed by measuring (1) lung hydroxyproline content as an index of collagen accumulation, (2) airway dysfunction by whole body plethysmography, and (3) histopathology. Bleomycin produced a 20% loss in body weight that was only 10% in the bleomycin/MnTBAP group. Bleomycin produced a twofold increase in hydroxyproline content that was decreased 23% by MnTBAP. Bleomycin produced a twofold increase in airway dysfunction that was also attenuated 30% by MnTBAP. Histopathologic analysis of the lungs of mice treated with bleomycin demonstrated a severe fibrotic response that was attenuated 28% by MnTBAP. Future studies on the oxidant mechanisms that MnTBAP is affecting in this bleomycin model of lung fibrosis may shed light on potential new therapeutic approaches for treating interstitial lung diseases.

Dependence of excitotoxic neurodegeneration on mitochondrial aconitase inactivation.

Using the inactivation of mitochondrial and cytosolic aconitases as markers of compartment-specific superoxide (O2(-)) production, we show that oxygen-glucose deprivation (OGD) or excitotoxin exposure produce a time-dependent inactivation of mitochondrial, but not cytosolic, aconitase in cortical cultures. To determine if mitochondrial O2(-) production was an important determinant in neuronal death resulting from OGD, metalloporphyrins with varying superoxide dismutase (SOD) activity were tested for their ability to protect against mitochondrial aconitase inactivation and cell death. OGD-induced mitochondrial aconitase inactivation and cell death was inhibited by manganese tetrakis (4-benzoic acid) porphyrin (MnTBAP), manganese tetrakis (N-ethylpyridinium-2-yl) porphyrin (MnTE-2-PyP) and NMDA receptor antagonists. By contrast, NMDA- or kainate (KA)-induced mitochondrial aconitase inactivation and cell death was inhibited by MnTBAP, but not MnTE-2-PyP. Moreover, both MnTBAP and MnTE-2-PyP penetrated mitochondrial fractions of cortical cells. These data suggest that mitochondrial aconitase inactivation closely correlates with subsequent neuronal death following excitotoxicity produced by OGD or NMDA/KA exposure. Assessment of biological rather biochemical antioxidant activities better predicted neuroprotection by metalloporphyrins. Moreover, antioxidants that protect oxidant-sensitive mitochondrial targets such as aconitase may be useful as therapies for disease states involving excitotoxicity.

Antioxidant imbalance in the lungs of cystic fibrosis transmembrane conductance regulator protein mutant mice.

Recent studies suggest that the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein modulates epithelial reduced glutathione (GSH) transport and when defective creates an antioxidant imbalance. To test whether the CFTR protein modulates lung antioxidant defenses in vivo, epithelial lining fluid (ELF) and lung tissue from CFTR knockout (CFTR-KO) and wild-type (WT) mice were compared for GSH content and the activities of glutathione reductase, glutathione peroxidase, and gamma-glutamyltransferase. In the CFTR-KO mice, the ELF concentration of GSH was decreased (51%) compared with that in WT mice. The concentration of GSH in the lung tissue of CFTR-KO mice, however, was not significantly different from that in WT mice. The activities of glutathione reductase and glutathione peroxidase in the lung tissue of CFTR-KO mice were significantly increased compared with those in WT mice (48 and 28%, respectively). Tissue lipid and DNA oxidation were evaluated by measurement of thiobarbituric acid-reactive substances and 8-hydroxy-2'-deoxyguanosine, respectively. The levels of thiobarbituric acid-reactive substances and 8-hydroxy-2'-deoxyguanosine in the lung tissue of CFTR-KO mice were significantly increased compared with those in WT mice. These data support our hypothesis that a mutation in the CFTR gene can affect the antioxidant defenses in the lung and may contribute to the exaggerated inflammatory response observed in CF.

Neuroprotection from delayed postischemic administration of a metalloporphyrin catalytic antioxidant.

Reactive oxygen species contribute to ischemic brain injury. This study examined whether the porphyrin catalytic antioxidant manganese (III) meso-tetrakis (N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP(5+)) reduces oxidative stress and improves outcome from experimental cerebral ischemia. Rats that were subjected to 90 min focal ischemia and 7 d recovery were given MnTE-2-PyP(5+) (or vehicle) intracerebroventricularly 60 min before ischemia, or 5 or 90 min or 6 or 12 hr after reperfusion. Biomarkers of brain oxidative stress were measured at 4 hr after postischemic treatment (5 min or 6 hr). MnTE-2-PyP(5+), given 60 min before ischemia, improved neurologic scores and reduced total infarct size by 70%. MnTE-2-PyP(5+), given 5 or 90 min after reperfusion, reduced infarct size by 70-77% and had no effect on temperature. MnTE-2-PyP(5+) treatment 6 hr after ischemia reduced total infarct volume by 54% (vehicle, 131 +/- 60 mm(3); MnTE-2-PyP(5+), 300 ng, 60 +/- 68 mm(3)). Protection was observed in both cortex and caudoputamen, and neurologic scores were improved. No MnTE-2-PyP(5+) effect was observed if it was given 12 hr after ischemia. MnTE-2-PyP(5+) prevented mitochondrial aconitase inactivation and reduced 8-hydroxy-2'-deoxyguanosine formation when it was given 5 min or 6 hr after ischemia. In mice, MnTE-2-PyP(5+) reduced infarct size and improved neurologic scores when it was given intravenously 5 min after ischemia. There was no effect of 150 or 300 ng of MnTE-2-PyP(5+) pretreatment on selective neuronal necrosis resulting from 10 min forebrain ischemia and 5 d recovery in rats. Administration of a metalloporphyrin catalytic antioxidant had marked neuroprotective effects against focal ischemic insults when it was given up to 6 hr after ischemia. This was associated with decreased postischemic superoxide-mediated oxidative stress.

Manganese-porphyrin reactions with lipids and lipoproteins.

Manganese porphyrin complexes serve to catalytically scavenge superoxide, hydrogen peroxide, and peroxynitrite. Herein, reactions of manganese 5,10,15,20-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP(5+)) with lipids and lipid hydroperoxides (LOOH) are examined. In linoleic acid and human low-density lipoprotein (LDL), MnTE-2-PyP(5+) promotes oxidative reactions when biological reductants are not present. By redox cycling between Mn(+3) and Mn(+4) forms, MnTE-2-PyP(5+) initiates lipid peroxidation via decomposition of 13(S)hydroperoxyoctadecadienoic acid [13(S)HPODE], with a second-order rate constant of 8.9 x 10(3) M(-1)s(-1)and k(cat) = 0.32 s(-1). Studies of LDL oxidation demonstrate that: (i) MnTE-2-PyP(5+) can directly oxidize LDL, (ii) MnTE-2-PyP(5+) does not inhibit Cu-induced LDL oxidation, and (iii) MnTE-2-PyP(5+) plus a reductant partially inhibit lipid peroxidation. MnTE-2-PyP(5+) (1-5 microM) also significantly inhibits FeCl(3) plus ascorbate-induced lipid peroxidation of rat brain homogenate. In summary, MnTE-2-PyP(5+) initiates membrane lipid and lipoprotein oxidation in the absence of biological reductants, while MnTE-2-PyP(5+) inhibits lipid oxidation reactions initiated by other oxidants when reductants are present. It is proposed that, as the Mn(+3) resting redox state of MnTE-2-PyP(5+) becomes oxidized to the Mn(+4) redox state, LOOH is decomposed to byproducts that propagate lipid oxidation reactions. When the manganese of MnTE-2-PyP(5+) is reduced to the +2 state by biological reductants, antioxidant reactions of the metalloporphyrin are favored.

A catalytic antioxidant metalloporphyrin blocks hydrogen peroxide-induced mitochondrial DNA damage.

Reactive oxygen species (ROS) have been implicated as the cause of cumulative damage to DNA, proteins and lipids that can ultimately result in cell death. A common problem when measuring oxidative DNA damage has been the introduction of modifications in the native state of the molecule by many DNA isolation methods. We circumvented this problem by employing direct PCR (DPCR) of whole cell lysates. DPCR of mouse lung fibroblasts performed better than PCRs containing template acquired by phenol/chloroform extraction or a commercially available genomic DNA isolation kit. We investigated the direct use of whole cell preparations in the polymerase chain reaction (PCR) to detect hydrogen peroxide (H(2)O(2))-mediated DNA damage. We observed a concentration-dependent decrease in amplification efficiency of a 4.3 kb mitochondrial (mt)DNA target in H(2)O(2)-treated mouse lung fibroblasts (MLFs). At low doses the efficiency of amplification returns to control levels over 24 h. We detected no change in amplification efficiency of a plasmid control containing our mtDNA target under any of the culture conditions employed in these studies. Treatment of MLFs with the catalytic antioxidant manganese(III) meso -tetrakis(4-benzoic acid)porphyrin (MnTBAP) attenuates the effects of H(2)O(2)exposure. When quantitated with an external standard the use of DPCR in tandem with a PCR amplification efficiency assay provides a powerful approach to assess oxidative mtDNA damage.

A mechanism of paraquat toxicity involving nitric oxide synthase.

Paraquat (PQ) is a well described pneumotoxicant that produces toxicity by redox cycling with cellular diaphorases, thereby elevating intracellular levels of superoxide (O-(2)). NO synthase (NOS) has been shown to participate in PQ-induced lung injury. Current theory holds that NO reacts with O-(2) generated by PQ to produce the toxin peroxynitrite. We asked whether NOS might alternatively function as a PQ diaphorase and reexamined the question of whether NO/O-(2) reactions were toxic or protective. Here, we show that: (i) neuronal NOS has PQ diaphorase activity that inversely correlates with NO formation; (ii) PQ-induced endothelial cell toxicity is attenuated by inhibitors of NOS that prevent NADPH oxidation, but is not attenuated by those that do not; (iii) PQ inhibits endothelium-derived, but not NO-induced, relaxations of aortic rings; and (iv) PQ-induced cytotoxicity is potentiated in cytokine-activated macrophages in a manner that correlates with its ability to block NO formation. These data indicate that NOS is a PQ diaphorase and that toxicity of such redox-active compounds involves a loss of NO-related activity.

Metalloporphyrin class of therapeutic catalytic antioxidants.

Metalloporphyrins have emerged as a novel class of catalytic antioxidants that scavenge a wide range of reactive oxygen species (ROS) such as superoxide, peroxide, peroxynitrite and lipid peroxyl radicals. Factors such as the type of metal centre, redox potential and electrostatic charge of the compounds are recognized as important determinants of their antioxidant activity and potency. These concepts have guided the development of metalloporphyrins with specific activities greater than those of the native superoxide dismutases. Several compounds in this class have been shown to be efficacious in a variety of in vitro and in vivo oxidative stress models of human diseases.

Reactive oxygen species regulate activation-induced T cell apoptosis.

Reactive oxygen species (ROS) mediate apoptosis in a number of cell types. We studied the role that ROS play in activated T cell apoptosis by activating T cells in vivo and then culturing them for a short time. Activated T cells died independently of Fas and TNF alpha. Their death was characterized by rapid loss of mitochondrial transmembrane potential (delta psi(m)), caspase-dependent DNA fragmentation, and superoxide generation. A superoxide dismutase mimetic, Mn (III) tetrakis (5, 10, 15, 20-benzoic acid) porphyrin (MnTBAP), protected T cells from superoxide generation, caspase-dependent DNA loss, loss of delta psi(m), and cell death. These results indicate that ROS can regulate signals involved in caspase activation and apoptosis and may contribute to peripheral T cell deletion.

Metalloporphyrins are potent inhibitors of lipid peroxidation.

The objectives of these studies were to determine whether metalloporphyrins could inhibit lipid peroxidation, characterize factors that influence their potency and compare their potency to prototypical antioxidants. Lipid peroxidation was initiated with iron and ascorbate in rat brain homogenates and the formation of thiobarbituric acid reactive species was used as an index of lipid peroxidation. Metalloporphyrins were found to be a novel and potent class of lipid peroxidation inhibitors. Inhibition of lipid peroxidation by metalloporphyrins was dependent on the transition metal ligated to the porphyrin, indicating that metal centered redox chemistry was important to the mechanism of their antioxidant activities. Manganese porphyrins with the highest superoxide dismutase (SOD) activities, MnOBTM-4-PyP and MnTM-2-PyP (charges are omitted throughout text for clarity), were the most potent inhibitors of lipid peroxidation with calculated IC50s of 1.3 and 1.0 microM, respectively. These manganese porphyrins were 2 orders of magnitude more potent than either trolox (IC50 = 204 microM) or rutin (IC50 = 112 microM). The potencies of the manganese porphyrins were related not only to their redox potentials and SOD activities, but also to other factors that may contribute to their ability to act as electron acceptors. The broad array of antioxidant activities possessed by metalloporphyrins make them attractive therapeutic agents in disease states that involve the overproduction of reactive oxygen species.

A novel neurological phenotype in mice lacking mitochondrial manganese superoxide dismutase.

Reactive oxygen species (ROS) have been implicated in a wide range of degenerative processes including amyotrophic lateral sclerosis, ischemic heart disease, Alzheimer disease, Parkinson disease and aging. ROS are generated by mitochondria as the toxic by-products of oxidative phosphorylation, their energy generating pathway. Genetic inactivation of the mitochondrial form of superoxide dismutase in mice results in dilated cardiomyopathy, hepatic lipid accumulation and early neonatal death. We report that treatment with the superoxide dismutase (SOD) mimetic Manganese 5, 10, 15, 20-tetrakis (4-benzoic acid) porphyrin (MnTBAP) rescues these Sod2tm1Cje(-/-) mutant mice from this systemic pathology and dramatically prolongs their survival. The animals instead develop a pronounced movement disorder progressing to total debilitation by three weeks of age. Neuropathologic evaluation reveals a striking spongiform degeneration of the cortex and specific brain stem nuclei associated with gliosis and intramyelinic vacuolization similar to that observed in cytotoxic edema and disorders associated with mitochondrial abnormalities such as Leighs disease and Canavans disease. We believe that due to the failure of MnTBAP to cross the blood brain barrier progressive neuropathology is caused by excessive mitochondrial production of ROS. Consequently, MnTBAP-treated Sod2tm1Cje(-/-) mice may provide an excellent model for examining the relationship between free radicals and neurodegenerative diseases and for screening new drugs to treat these disorders.

Manganic porphyrins possess catalase activity and protect endothelial cells against hydrogen peroxide-mediated injury.

Manganic porphyrins are redox active metal complexes that have been employed as superoxide dismutase mimics. We tested whether these metalloporphyrins could also dismute hydrogen peroxide (H2O2) and whether they could protect endothelial cells against H2O2. Both of the manganic metalloporphyrins tested were found to catalytically dismute H2O2. These manganic porphyrins also protected endothelial cells in dose-dependent manners against H2O2-mediated injury with MnTMPyP having an EC50 of 8 microM and MnTBAP having an EC50 of 15 microM. The zinc containing analogs of these porphyrins were inactive in dismuting H2O2 and did not protect. These studies further define the antioxidant capacity of metalloporphyrins in converting superoxide to H2O2 and H2O2 to water. These data suggest that manganic porphyrins may be useful therapeutics against disease states associated with the overproduction of reactive oxygen species.

Extracellular superoxide dismutase is upregulated with inducible nitric oxide synthase after NF-kappa B activation.

Inflammatory cytokines have been shown to upregulate secretion of the antioxidant enzyme extracellular superoxide dismutase (EC-SOD) in dermal fibroblasts and, in other cells, to stimulate production of nitric oxide (.NO). Because superoxide rapidly scavenges .NO, forming the injurious peroxynitrite anion (OONO-), we hypothesize that stimulated cells upregulate EC-SOD expression concurrently with .NO release. To test for coregulation of EC-SOD and .NO within the same cell, the timing of inducible nitric oxide synthase (iNOS) and EC-SOD transcription was measured after exposure of a rate type II pneumocyte analog, the L2 cell line, to a combination of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). Upregulation of iNOS and EC-SOD transcription occurred after 6 h of exposure, and transcription of both genes was linked by activation of the transcription factor nuclear factor-kappa B. Both EC-SOD and iNOS were elevated in rat lung homogenates 24 h after intratracheal instillation with IFN-gamma and TNF-alpha. The observation that EC-SOD and iNOS are temporally coregulated after cytokine exposure suggests the possibility of a critical mechanism by which cells might protect .NO and avoid the formation of OONO- during inflammation.

Diethyl dithiocarbamate-induced decomposition of S-nitrosothiols.

Diethyl dithiocarbamate (DDC) has been used extensively as an inhibitor of CuZn superoxide dismutase (SOD) in the study of superoxide and nitric oxide. Addition of DDC to solutions of the endogenous NO adduct S-nitrosoglutathione (GSNO) causes a rapid decrease in GSNO with concomitant formation of nitrite, nitrate, disulfuram, oxidized glutathione, and mixed disulfide. Nitric oxide and superoxide appear to be produced in the process. Product formation is best explained by a radical mechanism in which S-nitrosation of DDC facilitates disulfide formation following homolytic cleavage. S-Nitrosocysteine and S-nitroso-N-acetylpenicillamine are likewise unstable in the presence of DDC. These findings may complicate interpretation of experiments in which DDC is used to alter NO-mediated responses. Some biological actions of DDC may result from SNO elimination rather than SOD inactivation. Moreover, apparent DDC-induced potentiation of superoxide effects may derive from O2- produced during the conversion of SNO to NO.