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PubMed1993

Hyperoxic sheep pulmonary microvascular endothelial cells generate free radicals via mitochondrial electron transport.

Free radical generation by hyperoxic endothelial cells was studied using electron paramagnetic resonance (EPR) spectroscopy and the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the radical species produced, whether mitochondrial electron transport was involved, and the effect of the radical generation on cell mortality. Sheep pulmonary microvascular endothelial cell suspensions exposed to 100% O2 for 30 min exhibited prominent DMPO-OH and, occasionally, additional smaller DMPO-R signals thought to arise from the trapping of superoxide anion (O2-.), hydroxyl (.OH), and alkyl (.R) radicals. Superoxide dismutase (SOD) quenched both signals suggesting that the observed radicals were derived from O2-.. Studies with deferoxamine suggested that the generation of .R occurred secondary to the formation of .OH from O2-. via an iron-mediated Fenton reaction. Blocking mitochondrial electron transport with rotenone (20 microM) markedly decreased radical generation. Cell mortality increased slightly in oxygen-exposed cells. This increase was not significantly altered by SOD or deferoxamine, nor was it different from the mortality observed in air-exposed cells. These results suggest that endothelial cells exposed to hyperoxia for 30 min produce free radicals via mitochondrial electron transport, but under the conditions of these experiments, this radical generation did not appear cause cell death.

Animals↗

PubMed2004

Thalassemia.

New developments in the epidemiology, treatment and prognosis of thalassemia have dramatically altered the approach to the care of affected patients, and these developments are likely to have an even greater impact in the next few years. Demographic changes have required an awareness and understanding of the unique features of thalassemia disorders that were previously uncommon in North America but are now seen more frequently in children and recognized more consistently in adults. New methods for measuring tissue iron accumulation and new drugs to remove excessive iron are advancing two of the most challenging areas in the management of thalassemia as well as other transfusion-dependent disorders. Improved survival of patients with thalassemia has given new importance to adult complications such as endocrinopathies and hepatitis that have a major impact on the quality of life. This chapter describes how these changes are redefining the clinical management of thalassemia. In Section I, Dr. Renzo Galanello describes recent advances in iron chelation therapy. Several new chelators are either licensed in some countries, are in clinical trials or are in the late stages of preclinical development. Some of these iron chelators, such as deferiprone (DFP) and ICL670, are orally active. Others, such as hydroxybenzyl-ethylenediamine-diacetic acid (HBED) and starch deferoxamine, require parenteral administration but may be effective with less frequent administration than is currently required for deferoxamine. Chelation therapy employing two chelators offers the possibility of more effective removal of iron without compromising safety or compliance. Other strategies for chelation therapy may take advantage of the ability of particular chelators to remove iron from specific target organs such as the heart and the liver. In Section II, Dr. Dudley Pennell addresses cardiac iron overload, the most frequent cause of death from chronic transfusion therapy. The cardiac complications related to excessive iron may result from long-term iron deposition in vulnerable areas or may be due to the more immediate effects of nontransferrin-bound iron. Cardiac disease is reversible in some patients with intensive iron chelation therapy, but identification of cardiac problems prior to the onset of serious arrhythmias or congestive heart failure has proven difficult. New methods using magnetic resonance imaging (MRI) have recently been developed to assess cardiac iron loading, and studies suggest a clinically useful relationship between the results using these techniques and critical measures of cardiac function. Measurements such as T2* may help guide chelation therapy in individual patients and may also enhance the assessment of new chelators in clinical trials. The use of MRI-based technology also holds promise for wider application of non-invasive assessment of cardiac iron in the management of patients with thalassemia. In Section III, Dr. Melody Cunningham describes some of the important complications of thalassemia that are emerging as patients survive into adulthood. Hepatitis C infection is present in the majority of patients older than 25 years. However, antiviral therapy in patients with thalassemia has been held back by the absence of large clinical trials and concern about ribavirin-induced hemolysis. More aggressive approaches to the treatment of hepatitis C may be particularly valuable because of the additive risks for cirrhosis and hepatocellular carcinoma that are posed by infection and iron overload. Thrombosis is recognized with increasing frequency as a significant complication of thalassemia major and thalassemia intermedia, and pulmonary hypertension is now the focus of intense study. Risk factors for thrombosis such as splenectomy are being identified and new approaches to anticoagulation are being initiated. Pregnancies in women with thalassemia are increasingly common with and without hormonal therapy, and require a better understanding of the risks of iron overload and cardiac disease in the mother and exposure of the fetus to iron chelators. In Section IV, Dr. Elliott Vichinsky describes the dramatic changes in the epidemiology of thalassemia in North America. Hemoglobin E-beta thalassemia is seen with increasing frequency and poses a particular challenge because of the wide variability in clinical severity. Some affected patients may require little or no intervention, while others need chronic transfusion therapy and may be appropriate candidates for hematopoietic stem cell transplantation. Enhancers of fetal hemoglobin production may have a unique role in Hb E-beta thalassemia since a modest increase in hemoglobin level may confer substantial clinical benefits. Alpha thalassemia is also being recognized with increasing frequency in North America, and newborn screening for Hemoglobin Barts in some states is leading to early detection of Hb H disease and Hb H Constant Spring. New data clarify the importance of distinguishing these two disorders because of the increased severity associated with Hb H Constant Spring. The use of intrauterine transfusions to sustain the viability of fetuses with homozygous alpha thalassemia has created a new population of patients with severe thalassemia and has raised new and complex issues in genetic counseling for parents with alpha thalassemia trait.

Heart↗

PubMed2006

Action of chelators in iron-loaded cardiac cells: Accessibility to intracellular labile iron and functional consequences.

Labile iron in hemosiderotic plasma and tissue are sources of iron toxicity. We compared the iron chelators deferoxamine, deferiprone, and deferasirox as scavengers of labile iron in plasma and cardiomyocytes at therapeutic concentrations. This comprised chelation of labile plasma iron (LPI) in samples from thalassemia patients; extraction of total cellular iron; accessing labile iron accumulated in organelles and preventing formation of reactive-oxidant species; and restoring impaired cardiac contractility. Neonatal rat cardiomyocytes were used for monitoring chelator extraction of LCI (labile cell iron) as 59Fe; assessing in situ cell iron chelation by epifluorescence microscope imaging using novel fluorescent sensors for iron and reactive oxygen species (ROS) selectively targeted to organelles, and monitoring contractility by time-lapse microscopy. At plasma concentrations attained therapeutically, all 3 chelators eliminated LPI but the orally active chelators rapidly gained access to the LCI pools of cardiomyocytes, bound labile iron, attenuated ROS formation, extracted accumulated iron, and restored contractility impaired by iron overload. The effect of deferoxamine at therapeutically relevant concentrations was primarily by elimination of LPI. The rapid accessibility of the oral chelators deferasirox and deferiprone to intracellular labile iron compartments renders them potentially efficacious for protection from and possibly reversal of cardiac damage induced by iron overload.

Animals↗

PubMed1995

Survival in a case of massive paraquat ingestion.

Serious exposure to the herbicide paraquat usually results in death, either due to gastrointestinal caustic lesions, shock, and acute respiratory distress syndrome or related to the progressive development of pulmonary fibrosis associated with refractory hypoxemia. We report a case of suicidal paraquat ingestion in a 59-year-old man. Most of the indicators of poor prognosis were encountered in this patient. Treatment consisted of early digestive decontamination and hemodialysis, followed by antioxidant therapy, including the administration of deferoxamine (100 mg/kg in 24 h) and a continuous infusion of acetylcysteine (300 mg/kg/d during 3 weeks). The patient only developed a nonoliguric acute renal failure, a mild alteration of liver tests, and an impairment of CO transfer factor without any respiratory complaint. Renal and hepatic disturbances completely resolved within 1 month, whereas CO transfer factor remained altered 14 months later. This observation suggests that early administration of an antioxidant therapy, including deferoxamine and acetylcysteine could be usefully associated with measures that prevent digestive absorption or enhance elimination to limit systemic toxicity in potentially fatal paraquat poisoning.

Humans↗

PubMed1997

Hyperoxia exacerbates microvascular lung injury following acid aspiration.

OBJECTIVES: To examine the effects of an increase in ambient oxygen (O2) concentrations on the extent of inflammatory pulmonary damage following acid aspiration. DESIGN: Prospective, controlled laboratory study. SETTINGS: University-affiliated animal research facility. SUBJECTS: Male, Long Evans rats weighing 250 to 300 g. INTERVENTION: Rats were injured by instillation of 1.2 mL/kg normal saline solution/HCl, pH= 1.25 (acid), into the lungs via a tracheotomy. Animals were allowed to awaken and were exposed to 21%, 50%, or 98% O2 for 0 to 5 h (n/group > or = 10). In a separate set of experiments, injured rats exposed to 98% O2 were treated with different doses of deferoxamine, just prior to injury. Uninjured rats and rats injured with normal saline solution, pH = 5.3, were used as the control group. MEASUREMENTS: Injury was determined by assessing lung function (lung compliance and arterial blood gases) and alveolar-capillary wall integrity (wet/dry weight, lung albumin permeability index [PI], and intrapulmonary hemorrhage [HI]). RESULTS: Intrapulmonary instillation of acid increased PI, HI, and decreased static lung compliance compared to uninjured control animals. Increased ambient oxygen following acid aspiration decreased lung compliance, 1.06+/-0.03 mL/kg/cm H2O, in oxygen-exposed lungs when compared to the lungs exposed to air, 1.26+/-0.04, following a low pH aspirate (p<0.05). An increase in protein leakage into the lung tissue was noted in oxygen-exposed animals, PI=1.33+/-0.10, vs air-exposed rats, 0.89+/-0.07 (p<0.05). The hyperoxia-induced increase in lung injury was prevented by 30 mg/kg or higher deferoxamine treatment, 0.78+/-0.05 (p<0.05). Exposure of animals to 98% O2 for 2 h was sufficient to produce the same increase in microvascular protein leakage as 5-h exposure to O2 following low pH aspirate. CONCLUSION: Hyperoxia increases acid aspiration-induced inflammatory microvascular lung injury. This appears to be mediated by production of reactive species of O2.

Animals↗

PubMed2002

Alleviation of the two-cell block of ICR mouse embryos by polyaminocarboxylate metal chelators.

The present study was undertaken to examine the effects of various transition metal ion chelators, both polyaminocarboxylates (including nitrilotriacetate (NTA), ethylenediaminediacetate (EDDA), ethyleneglycolbistetraacetate (EGTA), ethylenediaminetetraacetate (EDTA) and diethylenetriaminepentaacetate (DTPA)) and non-polyaminocarboxylates (dipicolinic acid and deferoxamine), on the development in vitro of one-cell ICR strain mouse embryos to the four-cell and blastocyst stages. The order of stability constants of polyaminocarboxylates for transition metal ions such as zinc, copper and iron is as follows: NTA < or = EDDA < EGTA < EDTA < DTPA. Addition of 10 or 100 micromol polyaminocarboxylates x l(-1) to the medium significantly enhanced the development of most one-cell embryos (66-88%) beyond the two-cell stage compared with that (< 25%) in medium without polyaminocarboxylates. Although EDDA, EDTA and DTPA at 10 micromol x l(-1) induced the development of most one-cell embryos to the four-cell stage and beyond, a higher concentration (100 micromol x l(-1)) of NTA and EGTA was required to obtain a similar result. Therefore, the ability of polyaminocarboxylates to overcome the two-cell block is not correlated with their potency to chelate transition metal ions. In contrast, the non-polyaminocarboxylates dipicolinic acid and deferoxamine, at 10 and 100 micromol x l(-1), did not have the same effect. Taken together, the results indicate that the ability of polyaminocarboxylates to overcome the two-cell block in embryo development is due to some common feature or features other than the ability to chelate transition metal ions.

Animals↗

PubMed2005

Iron gathering of opportunistic pathogenic fungi. A mini review.

Iron is an essential nutrient for most organisms because it serves as a catalytic cofactor in oxidation-reduction reactions. Iron is rather unavailable because it occurs in its insoluble ferric form in oxides and hydroxides, while in serum of mammalian hosts is highly bound to carrier proteins such as transferrin, so the free iron concentration is extremely low insufficient for microbial growth. Therefore, many organisms have developed different iron-scavenging systems for solubilizing ferric iron and transporting it into cells across the fungal membrane. There are three major mechanisms by which fungi can obtain iron from the host: (a) utilization of a high affinity iron permease to transport iron intracellularly, (b) production and secretion of low molecular weight iron-specific chelators (siderophores), (c) utilization of a hem oxygenase to acquire iron from hemin. Patients with elevated levels of available serum iron treated with iron chelator, deferoxamine to remedy iron overload conditions have an increased susceptibility of invasive zygomycosis. Presumably deferoxamine predisposes patients to Zygomycetes infections by acting as a siderophore]. The frequency of zygomycosis is increasing in recent years and these infections respond very poorly to currently available antifungal agents, so new approaches to develop strategies to prevent and treat zygomycosis are urgently needed. Siderophores and iron-transport proteins have been suggested to function as virulence factors because the acquisition of iron is a crucial pathogenetic event. Biosynthesis and uptake of siderophores represent possible targets for antifungal therapy.

Biological Transport↗

PubMed2004

The design and development of deferiprone (L1) and other iron chelators for clinical use: targeting methods and application prospects.

Iron is essential for all human cells as well as neoplastic cells and invading microbes. Natural and synthetic iron chelators could affect biological processes involving iron and other metal ions in health and disease states. Iron overload is the most common metal toxicity condition worldwide. There are currently two iron chelating drugs, which are mostly used for the treatment of thalassaemia and other conditions of transfusional iron overload. Deferoxamine was until recently the only approved iron chelating drug, which is effective but very expensive and administered parenterally resulting in low compliance. Deferiprone (L1 or 1,2-dimethyl-3-hydroxypyrid-4-one) is the world's first and only orally active iron chelating drug, which is effective and inexpensive to synthesise thus increasing the prospects of making it available to most thalassaemia patients in third world countries who are not currently receiving any form of chelation therapy. Deferiprone has equivalent iron removal efficacy and comparable toxicity to deferoxamine. There are at least four other known iron chelators, which are currently being developed. Even if successful, these are not expected to become available for clinical use in the next five years and to be as inexpensive as deferiprone. The variation in the chemical, biological, pharmacological, toxicological and other properties of the chelating drugs and experimental chelators provide evidence of the difference in the mode of action of chelators and the need to identify and select molecular structures and substituents based on structure/activity correlations for specific pharmacological activity. Such information may increase the prospects of designing new chelating drugs, which could be targeted and act on different tissues, organs, proteins and iron pools that play important role not only in the treatment of iron overload but also in other diseases of iron and other metal imbalace and toxicity including free radical damage. Chelating drugs could also be designed, which could modify the enzymatic activity of iron and other metal containing enzymes, some of which play a key role in many diseases such as cancer, inflammation and atherosclerosis. Other applications of iron chelating drugs could involve the detoxification of toxic metals with similar metabolic pathways to iron such as Al, Cu, Ga, In, U and Pu.

Animals↗

PubMed2005

Enhanced oxidative stress by L-ascorbic acid within cells challenged by hydrogen peroxide.

It has been amply documented that L-ascorbic acid added to the medium of a cell culture increases oxidative damage, and this effect of L-ascorbic acid has been ascribed to the generation of reactive oxygen intermediates in the medium during its auto-oxidation. We have here questioned whether such an effect is exerted inside the cell as well, and if so, what its mechanism is. To assess thiol oxidation in the cell, we manipulated CHO cells so that they could express bacterial alkaline phosphatase in the cytoplasm. Alkaline phosphatase activity, which requires the formation of intramolecular disulfide bridges, was shown to appear when the cells were exposed to H2O2. This H2O2-induced activity increased more than 1.5 fold when L-ascorbic acid had been loaded in the cells by incubation with L-ascorbic acid-2-O-phosphate. Similar enhancing effects were also observed by assessing oxidation of glutathione, formation of protein carbonyls, and generation of reactive oxygen intermediates. Interestingly, the effects by the L-ascorbic acid-2-O-phosphate treatment were totally suppressed by addition of the membrane-permeable chelator deferoxamine to the medium, indicating the involvement of iron ions. Because the apoprotein of conalbumin, which binds iron ions with a high affinity, had no effect and because the same deferoxamine effect was observed with the cells incubated in balanced salt solution with no metal salts added, it was concluded that L-ascorbic acid acts as a pro-oxidant within the cell suffering oxidative stress, and that this effect is elicited through increased redox-cycling of iron in combination with L-ascorbic acid.

Alkaline Phosphatase↗

PubMed1996

The synergistic effect of elastase and hydrogen peroxide on vascular endothelial cell injury is due to the production of hydroxylradical in the endothelial cells.

Protease inhibitors such as aprotinin and urinastatin inhibited vascular endothelial cell injury induced by PMA-stimulated leukocytes, although their inhibitors did not suppress the production of active oxygen species released from leukocytes. On the other hand, in the presence of pancreas elastase (10 micrograms/ml), hydrogen peroxide (50 microM) caused severe injury of endothelial cells isolated from the bovine carotid artery (% specific 51Cr release, % SR = 42.9 +/- 3.3%), although the % SR elicited by elastase or hydrogen peroxide alone, respectively, was below 1%. Elastase and hydrogen peroxide acted synergistically on the injury of endothelial cells from the bovine carotid artery similarly to that in the endothelial cells isolated from the bovine coronary artery and human umbilical vein. Furthermore, elastase derived from both pancreas and leukocyte induced this synergistic action on endothelial cell injury. To clarify the mechanism of vascular endothelial cell injury induced by the combination of elastase and hydrogen peroxide, we examined the effects of various radical scavengers and protease inhibitors. Deferoxamine mesylate completely inhibited the endothelial cell injury, while protease inhibitors such as antitrypsin and macroglobulin had a protective effect. Pretreatment of endothelial cells with deferoxamine mesylate also protected against this cytotoxicity. These findings suggested that the synergistic effect of elastase and hydrogen peroxide on the endothelial cell injury is due to the production of hydroxylradical in the endothelium and that this synergistic action might be partially involved in the endothelial cell injury induced by activated leukocytes.

Animals↗

PubMed2001

Variability of biological responses to silicas: effect of origin, crystallinity, and state of surface on generation of reactive oxygen species and morphological transformation of mammalian cells.

Variously modified quartz dusts and one amorphous diatomaceous earth have been compared in their potential to release HO* radicals and in their activity in the Syrian hamster embryo (SHE) cell transformation assay. Both original dusts, made up by well-crystallized quartz particles, or by mostly amorphous, variously shaped, silica particles, were active in HO* release, were cytotoxic, and induced morphological transformation in SHE cells. The cristobalite dust, obtained by heating quartz above the phase transition temperature, lost any activity in free radical release, cytotoxicity, and transforming potency. Surface-modified quartz dusts were obtained by a mild etching with HF, by depriving the surface of trace iron with deferoxamine, or by enriching it with iron. The chemical and biological activity decreased in all cases. Both iron-deprived and iron-enriched quartz were nearly inactive. A linear correlation was found between the amount of HO* released by the particles and the transformation frequency. When the SHE cell assay was performed in the presence of mannitol or antioxidant enzymes (superoxide dismutase [SOD] or catalase), the number of transformed cells markedly decreased. This effect was more pronounced for catalase and mannitol than for SOD. HO* release was reduced, but not suppressed, by deferoxamine. All the above results are consistent with the presence of two kinds of surface sites active in HO* release and cell transformation: (1) silicon-based radicals, abundant on freshly ground dusts, which generate the HO* radicals without the superoxide ion as intermediate; and (2) isolated iron centers where the Haber-Weiss cycle takes place, with the superoxide ion as intermediate. The activities of both sites are inhibited by mannitol or catalase, whereas only the last one is inhibited by SOD.

Air Pollutants, Occupational↗

PubMed2000

Deferiprone: new preparation. Poorly assessed.

Deferiprone, an oral iron chelator, has been licensed in Europe for the treatment of iron overload in patients with beta thalassaemia who cannot be treated with deferoxamine because of adverse effects or the difficult administration schedule. (2) The clinical dossier is thin and methodologically mediocre. Even the preclinical assessment dossier is insufficient (there are no studies of carcinogenicity or hepatotoxicity). (3) The only available comparative trial, involving a small number of patients, suggests that deferiprone is less effective than deferoxamine in reducing ferritin levels and hepatic iron stores. (4) A non comparative trial suggests that long-term deferiprone therapy would not only fail, but would also increase the risk of liver fibrosis in some patients. This doubt is not dispelled by the other available non-comparative trials. (5) Deferiprone carries a risk of agranulocytosis in an estimated 1.2% of patients. (6) Deferiprone is teratogenic in two animal species.

Agranulocytosis↗

PubMed1992

Iron assimilation in Cryptococcus neoformans.

We studied the effects of iron chelators and of a thallium salt on growth of Cryptococcus neoformans in defined medium. An oxidant-sensitive mutant strain was found to require exogenous ferric iron for growth. Using this strain, we found that the synthetic iron chelator, N-hydroxyethylenediamine triacetate (HEDTA), in several saturation states, stimulated growth as well as the comparably saturated siderophore deferoxamine. This non-specific result makes the existence of a cryptococcal ferrihydroxamate receptor doubtful. The catechols, caffeic acid, L-3, 4-dihydroxyphenylalanine, epinephrine, gallic acid, 3-hydroxytyramine (dopamine) and norepinephrine, were tested for growth stimulation in iron deprivation, under conditions in which deferoxamine was stimulatory. Catechols were found to be either neutral or inhibitory. The ferrous iron chelator, bathophenanthroline disulfonate (BPDS), inhibited growth strongly in the absence of exogenous iron, suggesting that ferric ion must be reduced before it can be internalized. Direct evidence of extracellular reduction was provided by accumulation of red-coloured ferrous-BPDS complex. The inhibition caused by BPDS was relieved by ferric HEDTA, even in the presence of 10-fold increased BPDS, suggesting a second, low-affinity, non-reductive iron uptake pathway. This inference was further supported by the observation that toxicity of the non-reducible ferric analogue, thallium (III), is relieved by iron repletion.

Biological Transport↗

PubMed2003

Clinical experience of acute ferric chloride poisoning.

Ferric chloride is both a corrosive acid and iron compound; reports of poisoning in humans are rare. A retrospective study was conducted to evaluate patients with ferric chloride exposure reported to Poison Control Center-Taipei Veterans General Hospital during 1990-2001. After exclusion of incomplete records, 16 patients with ferric chloride exposure were analyzed (9 male, 7 female aged 12 to 70 y). The exposures were occupational inhalation (18.7%), suicidal ingestion (56.3%), and accidental ingestion (25.0%). Major symptoms and signs were nausea/vomiting (68.8%), sore throat (68.8%), abdominal pain (37.5%), oral ulcers (37.5%), metabolic acidosis (25.0%), aspiration pneumonia (18.8%), respiratory failure (12.5%), diarrhea (12.5%), and hypotension (12.5%). The severity of poisonings were fatal 6.3%, severe 18.8%, moderate 31.2%, mild 37.5%, and asymptomatic 6.3%. Deferoxamine therapy was given in 9 hospitalized patients with good recovery; however the fatal case did not receive deferoxamine due to rapid deterioration and a late diagnosis. The serum iron level known in 7 cases ranged from 40 to 2440 microg/dL. Ingestion of ferric chloride may result in serious morbidity and mortality. Inappropriate labeling and storage lead to accidental swallowing or misdiagnosis. Early diagnosis is important, especially in seriously poisoned patients.

Acute Disease↗

PubMedDate not supplied

[Drug interference with the determination of urinary proteins with pyrocatechol violet Upro Vitros slides].

The purpose of this study is to investigate drug's interference on proteinuria's determination with pyorcatechol violet (Vitros technology). Five drugs were selected: gelatin based vascular replacement fluids (Plasmion), deferoxamine (Desféral), ceftriaxone (Rocéphine), ceftazidime (Fortum) and imipeneme (Tienam). To state precisely the role played by slide's technology, a liquid phase dosage was simultaneously made with pyrocatechol violet. No interference was notified with therapeutical range for four of them: gelatin, deferoxamine, ceftazidime and imipeneme. Slides technology prevents from gelatin's interference because the colorant is sensible to this drugs. On the other hand, positive interference was observed with ceftriaxone for urinary concentration upper than 0.75 g/L. This concentration is lower than that notified by Vitros laboratory in the technical data.

Benzenesulfonates↗

PubMed1992

Reactive oxygen species formation as a biomarker of methylmercury and trimethyltin neurotoxicity.

Reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, and hydroxyl radicals are believed to be initiators of peroxidative cell damage. This paper focused on the use of 2',7'-dichlorofluorescein-diacetate (DCFH-DA) to quantitate cerebral ROS as an index for neurotoxicity. This technique employs an assay of dichlorofluorescein (DCF), the fluorescent product of dichlorofluorescein (DCFH). Data from studies using various free radical generating systems, several iron chelators and hydroxyl radical scavengers suggest that DCFH oxidation may result in several reactive intermediates. In a biological system (synaptosomes isolated from untreated rats) DCF fluorescence was stimulated by ascorbate or FeSO4, while deferoxamine inhibited the ascorbate/FeSO4-induced stimulation of DCF formation. Two organometals, methylmercury (MeHg) and trimethyltin (TMT), known to produce neurotoxicity were tested. In vitro exposure to MeHg (10-20 microM) increased the rate of formation of ROS while TMT (5-40 microM) had no effect. In vivo, 48 hr and 1 week after a single injection of MeHg (1 mg/kg, i.p.) in mice and 1 week after a single injection of MeHg (5 mg/kg, i.p.) in rats, the rate of formation of ROS in both rat and mouse cerebellum was significantly increased. Pretreatment with deferoxamine, a potent iron-chelator, prevented MeHg-induced increase of ROS. In hippocampus and frontal cortex, ROS formation rates were also elevated 48 hr after TMT injection (3 mg/kg, i.p.) in mice. These results demonstrate that DCF fluorescence provides a good measure of overall ROS formation in synaptosomes of both in vitro as well as in vivo systems. Since ROS formation was selectively increased in areas known to be specifically vulnerable to organometals (cerebellum in the case of MeHg and hippocampus in the case of TMT), these studies further support that oxidative damage may be the primary mechanism underlying the neurotoxicity induced by these organometals.

Animals↗

PubMed1992

[Role of neutrophil and hydroxyl radical in shock-induced gut origin infection].

The relative roles of hydroxyl radical and neutrophils in the pathogenesis of shock-induced mucosal injury and gut origin infection (GOI) were determined. The incidence of GOI was higher in the shocked rats (30 mmHg for 30 min) than the sham-shock controls (87% vs 12.5%; P less than 0.01). Administration of the hydroxyl radical scavenger, dimethyl sulfoxide (DMSO) or iron chelator and deferoxamine reduced the incidence of GOI from 87% to 20% and 40% respectively (P less than 0.05). DMSO and deferoxamine appeared to prevent shock-induced GOI by blunting the magnitude of shock-induced mucosal injury. In contrast, neutrophil depletion did not prevent GOI or protect the intestinal mucosal in the shocked rats. Instead, the incidence of systemic spread of bacteria past the mesenteric lymph nodes to the livers and spleens of the shocked rats was higher in the neutrophil depleted rats (56%) than any other group (7%) (P less than 0.01). Thus, shock-induced GOI and intestinal injury appears to be mediated by xanthine oxidase generated oxidants such as hydroxyl radical rather than neutrophil-generated factors. In addition, neutrophil depletion may be clinically deleterious, since it promotes systemic sepsis rather than preventing shock-induced GOI.

Animals↗

PubMed1992

Regulation of synthesis of p34cdc2 and its homologues and their relationship to p110Rb phosphorylation during cell cycle progression of normal human T cells.

In yeast, the protein kinase p34cdc2 plays a role in regulating both the G2 to M and G1 to S phase transitions. The discovery of multiple homologues of the protein in cells of higher eukaryotic organisms suggests that different cell cycle regulatory events may be performed by different kinases in such cells. Here, the synthesis and metabolism of the human forms of these proteins are described in a normal human cell type, peripheral blood T lymphocytes that have been stimulated to enter the cell cycle in vitro. Using a carboxyl-terminus antiserum specific for true p34cdc2, the protein could first be found in T cells at about 24 to 30 h after stimulation, just before the initiation of DNA synthesis. Three forms of the enzyme could be resolved by denaturing gel electrophoresis: an unphosphorylated form with an apparent molecular mass of 34,500 daltons and two phosphorylated derivatives. In cells synchronized at G2/M phase with nocodazole, p34 was almost entirely in the unphosphorylated form whereas the phosphorylated derivatives were more predominant in cultures arrested at the G1/S border with aphidicolin. The relationship of p34 synthesis to the phosphorylation of p110Rb, an event known to be associated with passage through late G1 and/or the G1/S phase transition, was also investigated. It was noted that p110Rb phosphorylation began before p34 synthesis first became detectable. Furthermore, it appeared that the two events could be largely uncoupled by treating cells with deferoxamine (10 microM), an iron chelating agent that arrests T cells at a point in late G1 phase but substantially before the G1 to S phase transition. Under these conditions, p110Rb phosphorylation was almost completely accomplished in the absence of significant p34 synthesis, a finding that suggests that most or all of p110 phosphorylation is performed by kinases other than p34. Because of this observation, extracts were next examined for p34-like molecules using an antibody against the so-called PSTAIRE domain found in all cdc2 homologues identified to date. A species of protein with a mobility slightly less than true p34 was found, even in resting T cells. Upon stimulation, this protein increased slightly in amount, and a second protein with a mobility greater than p34, a putative p33cdk2, was seen. Not only was the appearance of these proteins not inhibited by deferoxamine but they accumulated in cultures treated with the drug, suggesting that p33, and not p34, may be the G1 phase kinase for p110Rb.(ABSTRACT TRUNCATED AT 400 WORDS)

CDC2 Protein Kinase↗