Application of sodium ferrate produced from industrial wastes for TOC removal of surface water.

This study aimed to investigate the effect of sodium ferrate synthesized from industrial effluents (SF-W) and that of synthetized from analytical grade chemicals (SF-O) on total organic carbon (TOC) removal from surface water. Response surface methodology (RSM) was used to optimize the operating variables such as pH, dosing rate, rapid mixing time, and gentle mixing speed on TOC removal. A TOC removal of 89.805% and 79.79% was observed for SF-O and SF-W, respectively. Ferrate as SF-O and SF-W demonstrated 26.67% and 8.51% more TOC removal at a lower dosage compared to conventional chemicals such as chlorine, ozone, poly aluminum chloride (PAC) and polyelectrolyte. The optimum conditions of the independent variables including sodium ferrate (SF-O and SF-W), pH, rapid mixing time and gentle mixing speed were found to be 1.54 mg/L and 2.68 mg/L, 8.5, 30 s at 120 rpm for coagulation followed by 20 min of gentle mixing. Economic analysis showed that the application of SF instead of conventional chemicals provides a significant reduction in operational costs by about 68%, mainly because of the reduction of chemicals and energy consumption.

Direct administration of interleukin-1 and interferon-gamma to rat pancreas leads to the in vivo production of nitric oxide and expression of inducible nitric oxide synthase and inducible cyclooxygenase.

INTRODUCTION: Proinflammatory cytokines may play a pivotal role in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). In vitro, the formation of nitric oxide (NO) catalyzed by inducible NO synthase (iNOS) has been shown to be involved in the cytotoxic effects of cytokines on pancreatic beta cells. Cytokines have also been shown to cause the expression of inducible cyclooxygenase (COX-2) in isolated islets. AIMS: To describe a novel in vivo model that allows investigation of the effects of direct cytokine administration to the pancreas. METHODOLOGY AND RESULTS: By using this method, we demonstrate that administration of interleukin-1beta and interferon-gamma to rat pancreas results in the generation of NO in the treated pancreata as detected by NO trapping and electron paramagnetic resonance spectroscopy. Beta cells were identified as the source of the formed NO. Reverse transcription and polymerase chain reaction analyses showed that administration of cytokines to the pancreas leads to the expression of iNOS and COX-2 mRNA in the pancreas tissue as well as the islets isolated from such tissues. The compound phenyl N-tert-butylnitrone, which protects mice against streptozotocin-induced IDDM, inhibits NO formation and downregulates both iNOS and COX-2 mRNA levels.

Inhibition of the cytokine-mediated inducible nitric oxide synthase expression in rat insulinoma cells by phenyl N-tert-butylnitrone.

Cytokines and nitric oxide (NO) have been implicated in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). We have shown that the spin-trapping agent phenyl N-tert-butylnitrone (PBN) protects against streptozotocin (STZ)-induced IDDM in mice. In order to gain more insights into the mechanism(s) of the protective action of PBN against IDDM, we have investigated the effect of this compound on the cytokine-induced NO generation (measured as nitrite) in rat insulinoma RIN-5F cells. Our results demonstrate that PBN cotreatment prevents the generation of nitrite by RIN-5F cells induced by treatment with tumor necrosis factor-alpha, interleukin 1beta, and interferon-gamma in a dose-dependent fashion. The generation of NO as a result of cytokine treatment and the inhibitory effect of PBN were further confirmed by electron paramagnetic resonance spectroscopy. Aminoguanidine, a selective inhibitor of inducible nitric oxide synthase (iNOS), abolished the cytokine-induced nitrite generation whereas N-nitro-l-arginine, an inhibitor more selective for other NOS isoforms, was significantly less effective. Western and Northern analyses demonstrated that PBN inhibits the cytokine-mediated expression of iNOS at the transcriptional level. Cytokine-induced nitrite formation was also inhibited by the two antioxidant agents alpha-lipoic acid and N-acetylcysteine. These results indicate that PBN protects against IDDM at least in part by prevention of cytokine-induced NO generation by pancreatic beta-cells.

COX-2 inhibition prevents insulin-dependent diabetes in low-dose streptozotocin-treated mice.

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease believed to be caused by an inflammatory process in the pancreas leading to selective destruction of the beta cells. Inducible cyclooxygenase (COX-2) is expressed under inflammatory conditions and its product prostaglandin E(2) (PGE(2)) is an important inflammation mediator. We report here that administration of the selective COX-2 inhibitor NS-398 prevents the onset of diabetes in mice brought on by multiple low-doses of streptozotocin (STZ). Histological observations indicated that STZ-mediated destruction of beta cells was prevented by NS-398 treatment. Delayed (day 3) administration of NS-398 was also protective in this model. No protective effect was observed when NS-398 was administered prior to a high, toxic dose of STZ. These results demonstrate the critical importance of COX-2 activity in autoimmune destruction of beta cells, and point to the fact that COX-2 inhibition can potentially develop into a preventive therapy against IDDM.

Dietary choline restriction causes complex I dysfunction and increased H(2)O(2) generation in liver mitochondria.

Removal of choline from the diet results in accumulation of triglycerides in the liver, and chronic dietary deficiency produces a non-genotoxic model of hepatocellular carcinoma. An early event in choline deficiency is the appearance of oxidized lipid, DNA and protein, suggesting that increased oxidative stress may facilitate neoplasia in the choline deficient liver. In this study, we find that mitochondria isolated from rats fed a choline-deficient, L-amino acid defined diet (CDAA) demonstrate impaired respiratory function, particularly in regard to complex I-linked (NADH-dependent) respiration. This impairment in mitochondrial electron transport occurs coincidentally with alterations in phosphatidylcholine metabolism as indicated by an increased ratio of long-chain to short-chain mitochondrial phosphatidylcholine. Moreover, hydrogen peroxide (H(2)O(2)) generation is significantly increased in mitochondria isolated from CDAA rats compared with mitochondrial from normal rats, and the NADH-specific yield of H(2)O(2) is increased by at least 2.5-fold. These findings suggest an explanation for the rapid onset of oxidative stress and energy compromise in the choline deficiency model of hepatocellular carcinoma and indicate that dietary choline withdrawal may be a useful paradigm for the study of mitochondrial pathophysiology in carcinogenesis.

Inhibition by phenyl N-tert-butyl nitrone of early phase carcinogenesis in the livers of rats fed a choline-deficient, L-amino acid-defined diet.

Male Wistar rats were fed a choline-deficient, L-amino acid-defined (CDAA) diet alone or in combination with a nitrone-based free radical trapping agent, phenyl N-tert-butyl nitrone (PBN) in the drinking water at the concentrations of 0.013, 0.065, and 0.130% for 12 weeks. PBN inhibited the changes that are normally induced in the livers of rats by the CDAA diet feeding, i.e., development of putative preneoplastic lesions, proliferation of connective tissue, reduction of glutathione S-transferase activity, formation of 8-hydroxyguanine in DNA, and an increase in inducible cyclo-oxygenase (COX2) activity. PBN, however, did not prevent the increases in the COX2 mRNA or protein levels brought on by the CDAA diet These results indicate that the loss of glutathione S-transferase activity and COX2 induction may play significant roles in rat liver carcinogenesis by the CDAA diet and that PBN prevents neoplasia not only by its radical scavenging activity but also by inhibiting COX2 activity at the catalytic level.

Potential mechanisms of photodynamic inactivation of virus by methylene blue. I. RNA-protein crosslinks and other oxidative lesions in Q beta bacteriophage.

A spectrum of oxidative lesions was observed in a bacteriophage-based model system that is very sensitive to the photodynamic activity of selected dyes. When suspensions of the intact bacteriophage Q beta were exposed to methylene blue plus light (MB + L), inactivating events, or "hits" occurred that were oxygen-dependent and that were associated with the formation of several specific lesions: (1) carbonyl moieties on proteins, (2) 8-oxo-7,8-dihydroguanine (8-oxoGua), and (3) single-strand breaks (ssb) in the RNA genome and (4) RNA-protein crosslinks. Formation of carbonyl groups associated with protein in the Q beta phage preparation correlated positively with photoinactivation of the phage with increasing doses of either of the sensitizers MB or rose bengal. Strand breaks in the Q beta genomic RNA were observable at high MB concentrations but appeared not to be significant at the lower concentrations of MB, as full-length Q beta RNA was observable well beyond the 99% inactivation point in MB dosage. It was shown that the number of 8-oxoGua lesions were unlikely to be sufficient to account for the number of lethal events. Following exposure to MB + L, crosslink formation between Q beta RNA and protein was observed by virtue of the location of RNA at the interface of phenol-aqueous extractions of phage suspensions. A significant increase over background of RNA-protein complexes (including full-length Q beta RNA) was observed at the lowest concentration of MB tested (0.5 microM), which corresponded roughly to an average of 2 lethal hits per phage or approximately 13% survival compared to the zero MB control (100% survival). Due to its close correlation with Q beta inactivation and its expected lethality, RNA-protein crosslink formation may be important as an inactivating lesion in bacteriophage Q beta following MB + L exposure.

Quantitation of protein-bound 3-nitrotyrosine and 3,4-dihydroxyphenylalanine by high-performance liquid chromatography with electrochemical array detection.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been implicated in myriad disease etiologies and may represent an obligate pathologic sequelus of inflammation. Unfortunately, few sensitive and specific analytical techniques exist for the routine assay of biomarkers indicative of ROS and RNS elaboration. In this study, high-performance liquid chromatography is used in conjunction with coulometric electrochemical array (HPLC-EC) detection to allow ultrasensitive determination of protein-bound 3-nitrotyrosine and 3, 4-dihydroxyphenylalanine (3-hydroxytyrosine) as specific in situ biomarkers of protein exposure to reactive nitrating and oxidizing species. Tyrosine and derivatives can be analyzed simultaneously with practical detection limits for tyrosine, 3-NT, and 3,4-Dopa being 10, 50, and 2 pmol, respectively, in as little as 20 microL of sample. HPLC-EC array detection allows two-dimensional resolution of chromatograms, greatly facilitating peak detection and confidence assignment. A method of sample preparation wherein tyrosine analogs are enzymatically hydrolyzed from protein without the need for sample extraction, concentration, or derivatization is reported.

Spin trapping agent phenyl N-tert-butylnitrone protects against the onset of drug-induced insulin-dependent diabetes mellitus.

Insulin-dependent diabetes mellitus is an autoimmune disease believed to be caused by an inflammatory process in the pancreas leading to selective destruction of the beta-cells. Cytokines and nitric oxide (NO) have been shown to be involved in this destruction. Phenyl N-tert-butylnitrone (PBN) has demonstrated protective effects against several pathological conditions including ischemia-reperfusion injury and endotoxin-induced shock. We report here that PBN co-administration can prevent the onset of the STZ-induced diabetes in mice. PBN co-treatment inhibited the streptozotocin (STZ)-induced hyperglycemia, the elevation in the level of glycated hemoglobin and weight loss in the treated mice. Histological observations indicated destruction of B-cells in the STZ-treated animals and its prevention by PBN co-treatment. EPR spin trapping experiments in the pancreas indicated the in vivo formation of NO in STZ-treated animals and its attenuation by PBN treatment.

Free radical production during ethanol intoxication, dependence, and withdrawal.

Indices of free radical production and cell damage were examined in male Sprague-Dawley rats chronically exposed to either ethanol (ETOH) or water vapor. In experiment 1, rats experienced either 1 or 11 cycles of ETOH exposure and withdrawal. Brain tissue was harvested 12 hr after ETOH exposure, and 1 hr after being injected with sodium salicylate as a scavenger. Brain tissue was analyzed for the formation of salicylate hydroxylation products as a measure of .OH production during withdrawal. Significant group differences for .OH production were demonstrated for 2,3- and 2,5-dihydroxybenzoic acid in the single cycle ETOH exposed rats compared with their water cohorts. A significant between group difference for 2,5-dihydroxybenzoic acid, only, was demonstrated for the multiple cycles of ETOH exposure. Spontaneous seizures were shown to correlate with increased production of .OH in ETOH exposed rats. In experiment 2, brain tissue was harvested from different groups of rats after removal from the chambers, at 0, 2, 12, 24, 36, and 48 hr after a single exposure cycle. Tissue was analyzed for (1) salicylate hydroxylation (as above), (2) glutamine synthetase activity, (3) whole brain glutamate concentration, and (4) oxidized protein. A multiple regression analysis was conducted on the five dependent variables and found they could be predicted by specific behavioral and neurological ratings. These data suggest that cell damage during withdrawal may have multiple time-dependent components.

Nitrone-based free radical traps as neuroprotective agents in cerebral ischaemia and other pathologies.

Nitrone-based spin trapping compounds have been shown to protect experimental animals from pathology associated with ischaemia/reperfusion injury, endotoxaemia, natural and accelerated aging, certain xenobiotics, and physical trauma. Moreover, these compounds have an intriguing nootropic action. Nitrones affect pathophysiological correlates in both the central nervous system and peripheral organ systems. These compounds have been shown to affect cellular oxidation state and oxidatively sensitive enzyme systems, but the precise mode of nitrone action has not been elucidated. Recent discoveries regarding the ability of nitrones to suppress gene transcriptional events associated with pathophysiological states, particularly the elaboration of NF kappa B-regulated cytokines and inducible nitric oxide synthase, argue that nitrones may act at a proximal level to oxidatively sensitive signal amplification systems.

Reactive oxygen species-mediated inactivation of pyruvate dehydrogenase.

Brain ischemia reperfusion causes increased formation of reactive oxygen species (ROS). Activity of the mitochondrial enzyme pyruvate dehydrogenase (PDH) has been shown to undergo a significant decrease following reperfusion of the ischemic tissue. We have examined the effect of a superoxide radical-generating system (xanthine oxidase/hypoxanthine, XO/HX) on the activity of this enzyme. Incubation of PDH in the presence of XO/HX resulted in its inactivation. The degree of the inactivation was dependent on the amount of XO present, which correlated linearly with the concentration of superoxide radical generated by this system. The activity of lactate dehydrogenase, an enzyme resistant to inactivation by ischemia reperfusion, was not affected by this system. Superoxide dismutase partially prevented and catalase exerted a nearly complete protective effect against the inactivation of PDH. Deferoxamine was partially protective. The sulfhydryl protective reagents, dithiothreitol and glutathione, prevented the inactivation of PDH, even though to varying degrees, which implicates sulfhydryl oxidation. A hydroxyl radical-generating system (hydrogen peroxide irradiated with ultraviolet radiation) effectively inactivated PDH. These results demonstrate that PDH is susceptible to damage and inactivation by ROS and point to the involvement of Fenton chemistry and hydroxyl radicals formed through it in PDH inactivation by XO/HX. A similar mechanism may be responsible for the PDH inactivation during ischemia/reperfusion.

Inactivation of glutathione peroxidase by benzaldehyde.

Chronic benzaldehyde exposure is known to cause central nervous system (CNS) disturbances. Previous studies have shown that benzaldehyde causes the formation of reactive oxygen species (ROS) in rat synaptosomal fractions. Benzaldehyde has also been implicated in ROS formation in the CNS of rats treated with toluene. We have found that benzaldehyde effectively inactivates the antioxidant enzyme glutathione peroxidase (Ki approximately 15 microM), but has no effect on the other antioxidant enzymes tested: catalase, superoxide dismutase, and glutathione reductase. This effect has been found to be specific to benzaldehyde since other structurally related and unrelated aldehydes tested were found to be devoid of inactivating capacity toward glutathione peroxidase. Since glutathione peroxidase is the main enzyme responsible for removal of hydrogen peroxide and organic hydroperoxides in brain, its inactivation by benzaldehyde may be a main contributor to the observed ROS formation and the observed neurotoxicity caused by either benzaldehyde or toluene exposure.

In vivo trapping of nitric oxide in the brain of neonatal rats treated with the HIV-1 envelope protein gp 120: protective effects of alpha-phenyl-tert-butylnitrone.

AIDS dementia complex is a neurological syndrome characterized by cognitive deficits and motor and behavioral dysfunction. The HIV-1 envelope glycoprotein gp 120 has been implicated in the development of AIDS dementia. This protein has been shown to be neurotoxic and to cause learning impairment and retardation of the development of complex motor behavior in rat neonates. Nitric oxide has been implicated in gp 120-induced neurotoxicity. In the present study, we report for the first time in vivo evidence for the formation of nitric oxide in the CNS as a result of multiple subcutaneous injections of gp 120 to neonatal rats. Nitric oxide was trapped in the brain of neonatal rats by N-methyl-D-glucamine dithiocarbamate-Fe and the nitric oxide content measured by electron paramagnetic resonance spectroscopy. The nitrone-based spin trap alpha-phenyl-tert-butylnitrone at 50 mg/kg was found to prevent gp 120-mediated nitric oxide formation and to also protect against gp 120-induced behavioral impairment.

Susceptibility of glutathione peroxidase and glutathione reductase to oxidative damage and the protective effect of spin trapping agents.

Susceptibility of two key protective enzymes, glutathione peroxidase (GPX) and glutathione reductase (GR), to oxidative damage and the possible protective action of spin traps have been studied. Several oxidizing protocols including: (a) Fe(II) or Fe(III)/ascorbate, (b) a singlet oxygen producing system (methylene blue and visible light), (c) ozone, and (d) a hydroxyl radical-generating system (hydrogen peroxide/uv light) have been employed. Our results show that both enzymes are susceptible to oxidative modification and damage as indicated by the loss of activity and formation of carbonyl groups (in the case of GR). Treatment of GR with any of the mentioned oxidants resulted in formation of carbonyl groups and inactivation except when treated with iron, where the observed carbonyl formation was not accompanied with significant activity loss. GPX was inactivated to varying degrees when treated with the mentioned oxidants, but no carbonyls were detected. Ultraviolet exposure per se resulted in inactivation of both enzymes. Presence of the spin traps N-tert-butyl-alpha-phenylnitrone or 5,5'-dimethyl-1-pyroline-N-oxide was effective in protecting the enzymes against oxidation by uv, hydrogen peroxide/uv, and ozone as determined by the preservation of activity and decreased carbonyl content. The degree of protection, however, was found to be specific for each enzyme and for the employed oxidizing system.

Further insights into the molecular mechanisms of action of the serotonergic neurotoxin 5,7-dihydroxytryptamine.

Autoxidation and various enzyme-mediated oxidations of the serotonergic neurotoxin 5,7-dihydroxytryptamine (1) give 5-hydroxytryptamine-4,7-dione (2) and 6,6'-bi(5-hydroxytryptamine-4,7-dione) (3) as the major products. When administered into the ventricular system of mice 2 and 3 are general toxins. The LD50 values for 2 (29.6 +/- 0.04 micrograms) and 3 (25.4 +/- 0.30 micrograms) are lower than that for 1 (51.8 +/- 0.28 micrograms). In the presence of cellular reductants (glutathione, cysteine, ascorbate) and molecular oxygen, or when incubated with rat brain homogenate, 2 and 3 redox cycle and form superoxide radical anion, O2.-, as a byproduct. The lethal effects of 2 and 3 when introduced into the brain may in part be due to such redox cycling reactions which deplete oxygen levels and, as a result of Haber-Weiss chemistry deriving from O2.-, form the cytotoxic hydroxyl radical (HO.). Intraventricular administration of 2 and 3 to mice causes only relatively minor and transient (ca. 1 h to 1 day) changes in whole brain levels of dopamine, 5-hydroxytryptamine (from both 2 and 3), acetylcholine, and choline (from 2 only). These changes differ from the profound and long-lasting serotonergic deficit evoked by 1. On the basis of these results a hypothesis has been formulated which proposes that the selective neurotoxicity of 1 derives from its rapid uptake into serotonergic neurons where it is oxidatively converted to 2 and 3. Redox cycling reactions of 2 and 3 then result in the depletion of intraneuronal oxygen and concomitant formation of O2.-. Dismutation of O2.- gives H2O2 which, as a result of transition metal ion-catalyzed Haber-Weiss chemistry, yields HO.. Thus, neuronal damage and death might result from the combined effects of hypoxia and HO. formation.

Chemical and enzyme-mediated oxidation of the serotonergic neurotoxin 5,7-dihydroxytryptamine: mechanistic insights.

The oxidation chemistry and biochemistry of the serotonergic neurotoxin 5,7-dihydroxytryptamine (1) has been studied under anaerobic and aerobic conditions in aqueous solution at physiological pH. Under anaerobic conditions, one-electron oxidants (ferricytochrome c, peroxidase/H2O2, ceruloplasmin, Cu2+) generate a radical intermediate. Dimerization of the C(6)-centered resonance form of this radical followed by secondary oxidations yields 3-(2-aminoethyl)-6-[3-(2-aminoethyl)-1,7-dihydro- 5-hydroxy-7-oxo-6H-indol-6-ylidene]-1-H-indole-5,7(4H,6H)-dione. Under aerobic conditions, molecular O2 attacks the C(4)-centered 1 radical to yield a hydroperoxy radical which decomposes to 5-hydroxytryptamine-4,7-dione (2). Autoxidation of 1 proceeds by primary attack by molecular O2 on a C(4)-centered carbanion to form a superoxide-radical complex. This rearranges to a C(4)-centered hydroperoxide which decomposes to 2. A C(6)-centered carbanion of 1 combines with 2 to give, ultimately, 6,6'-bi-5-hydroxytryptamine-4,7-dione (3). Trace concentrations of transition metal ions (Fe3+, Fe2+, Cu2+, Mn2+) catalyze the autoxidation of 1 by catalytic cycles in which a hydroperoxide intermediate plays key roles. A byproduct of the transition metal-catalyzed oxidation of 1 is superoxide, O2-. Because of its enormous basicity O2- facilitates deprotonation of 1. The C(4)-centered carbanion so produced is oxidized by molecular O2 or by the hydroperoxy radical (HO2) to give radical intermediates and thence 2 and 3. Mechanistic pathways leading to the various products of oxidation of 1 are proposed and the potential roles of oxidation reactions of the indolamine are related to its neurodegenerative properties.

Autoxidation of the serotonergic neurotoxin 5,7-dihydroxytryptamine.

The indolic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) has been widely speculated to express its neurodegenerative effects as a result of intraneuronol autoxidation. Until recently, it was believed that autoxidation led to reactive electrophilic quinone imine species which alkylated neuronal membrane proteins and that byproducts of the autoxidation reaction were cytotoxic reduced-oxygen species. This study reveals that at physiological pH carbanions of 5,7-DHT act as the primary electron-donor species to yield C(4)- and C(6)-centered free radical superoxide complexes in a 1:2 ratio. The C(4)-centered complex reacts to yield, ultimately, 5-hydroxytryptamine-4,7-dione which has been shown to be a significantly more powerful neurotoxin than 5,7-DHT. The C(6)-centered radical superoxide complexes react to give 6,6'-bis(5-hydroxytryptamine-4,7-dione). It is likely that the latter reaction yields O2.- as a cytotoxic byproduct.