A New Concept in Antidiabetic Therapeutics: A Concerted Removal of Labile Iron and Intracellular Deposition of Zinc.

BACKGRUOUND: The inflammatory process is known to be an integral part of the pathophysiology of type 2 diabetes mellitus (T2DM). The "labile," redox-active iron, serving as a catalyst in Fenton reaction, producing the deleterious reactive oxygen species, triggering and maintaining inflammation, is hypothesized to play a causative role in this process. Concenter Biopharma continued the development of a new platform of iron chelators (Zygosids), first initiated at the Hebrew University of Jerusalem, Israel (HUJI), acting via the novel mechanism, based on a sequestration of the labile redox-active iron and its substitution by zinc or gallium. The mode of action of Zygosids is based on the higher affinity of the metal-binding moiety of the complex to Fe3+ in comparison to already bound ion, leading to rapid release of the ion of another metal and chelation of Fe3+. Concomitantly, zinc ion, released by the complex, is known for its antidiabetic and anti-inflammatory role. METHODS: The therapeutic effect of zinc-desferrioxamine (Zygosid-50) and gallium-desferrioxamine, was tested on fat sand rat (Psammomys obesus) model of diet-induced T2DM and on Leprdb transgenic diabetic mice. RESULTS: Zygosids demonstrated an ability to noticeably reduce blood glucose and insulin levels and improve the lipid profile. Moreover, an ability to mitigate insulin resistance by >90% was shown on the sand rat model. In addition, a potent anti-inflammatory effect, expressed as a diminishment of the proinflammatory cytokines in tissue levels, was demonstrated. CONCLUSION: Zygosids demonstrated robust therapeutic efficacy in treatment of T2DM. Importantly, no adverse effects were detected, in all the experiments, indicating high safety profile.

Sevoflurane postconditioning is not mediated by ferritin accumulation and cannot be rescued by simvastatin in isolated streptozotocin-induced diabetic rat hearts.

Sevoflurane postconditioning (sevo postC) is an attractive and amenable approach that can protect the myocardium against ischemia/reperfusion (I/R)-injury. Unlike ischemic preconditioning (IPC), sevo postC does not require additional induced ischemic periods to a heart that is already at risk. IPC was previously shown to generate myocardial protection against I/R-injury through regulation of iron homeostasis and de novo ferritin synthesis, a process found to be impaired in the diabetic state. The current study investigated whether alterations in iron homeostasis and ferritin mRNA and protein accumulation are also involved in the cardioprotective effects generated by sevo postC. It was also investigated whether the protective effects of sevo postC in the diabetic state can be salvaged by simvastatin, through inducing nitric oxide (NO) bioavailability/activity, in isolated streptozotocin (STZ)-induced diabetic hearts (DH). Isolated rat hearts from healthy Controls and diabetic animals were retrogradely perfused using the Langendorff configuration and subjected to prolonged ischemia and reperfusion, with and without (2.4 and 3.6%) sevo postC and/or pre-treatment with simvastatin (0.5 mg/kg). Sevo postC significantly reduced infarct size and improved myocardial function in healthy Controls but not in isolated DH. The sevo postC mediated myocardial protection against I/R-injury was not associated with de novo ferrtin synthesis. Furthermore, simvastatin aggravated myocardial injury after sevo postC in STZ-induced DHs, likely due to increasing NO levels. Despite the known mechanistic overlaps between PC and postC stimuli, distinct differences underlie the cardioprotective interventions against myocardial I/R-injury and are impaired in the DH. Sevo postC mediated cardioprotection, unlike IPC, does not involve de novo ferritin accumulation and cannot be rescued by simvastatin in STZ-induced DHs.

Protection by Nitric Oxide Donors of Isolated Rat Hearts Is Associated with Activation of Redox Metabolism and Ferritin Accumulation.

Preconditioning (PC) procedures (ischemic or pharmacological) are powerful procedures used for attaining protection against prolonged ischemia and reperfusion (I/R) injury, in a variety of organs, including the heart. The detailed molecular mechanisms underlying the protection by PC are however, complex and only partially understood. Recently, an 'iron-based mechanism' (IBM), that includes de novo ferritin synthesis and accumulation, was proposed to explain the specific steps in cardioprotection generated by IPC. The current study investigated whether nitric oxide (NO), generated by exogenous NO-donors, could play a role in the observed IBM of cardioprotection by IPC. Therefore, three distinct NO-donors were investigated at different concentrations (1-10 µM): sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP). Isolated rat hearts were retrogradely perfused using the Langendorff configuration and subjected to prolonged ischemia and reperfusion with or without pretreatment by NO-donors. Hemodynamic parameters, infarct sizes and proteins of the methionine-centered redox cycle (MCRC) were analyzed, as well as cytosolic aconitase (CA) activity and ferritin protein levels. All NO-donors had significant effects on proteins involved in the MCRC system. Nonetheless, pretreatment with 10 µM SNAP was found to evoke the strongest effects on Msr activity, thioredoxin and thioredoxin reductase protein levels. These effects were accompanied with a significant reduction in infarct size, increased CA activity, and ferritin accumulation. Conversely, pretreatment with 2 µM SIN-1 increased infarct size and was associated with slightly lower ferritin protein levels. In conclusion, the abovementioned findings indicate that NO, depending on its bio-active redox form, can regulate iron metabolism and plays a role in the IBM of cardioprotection against reperfusion injury.

The Loss of Myocardial Benefit following Ischemic Preconditioning Is Associated with Dysregulation of Iron Homeostasis in Diet-Induced Diabetes.

Whether the diabetic heart benefits from ischemic preconditioning (IPC), similar to the non-diabetic heart, is a subject of controversy. We recently proposed new roles for iron and ferritin in IPC-protection in Type 1-like streptozotocin-induced diabetic rat heart. Here, we investigated iron homeostasis in Cohen diabetic sensitive rat (CDs) that develop hyperglycemia when fed on a high-sucrose/low-copper diet (HSD), but maintain normoglycemia on regular-diet (RD). Control Cohen-resistant rats (CDr) maintain normoglycemia on either diet. The IPC procedure improved the post-ischemic recovery of normoglycemic hearts (CDr-RD, CDr-HSD and CDs-RD). CDs-HSD hearts failed to show IPC-associated protection. The recovery of these CDs-HSD hearts following I/R (without prior IPC) was better than their RD controls. During IPC ferritin levels increased in normoglycemic hearts, and its level was maintained nearly constant during the subsequent prolonged ischemia, but decayed to its baseline level during the reperfusion phase. In CDs-HSD hearts the baseline levels of ferritin and ferritin-saturation with iron were notably higher than in the controls, and remained unchanged during the entire experiment. This unique and abnormal pattern of post-ischemic recovery of CDs-HSD hearts is associated with marked changes in myocardial iron homeostasis, and suggests that iron and iron-proteins play a causative role/s in the etiology of diabetes-associated cardiovascular disorders.

Zn/Ga-DFO iron-chelating complex attenuates the inflammatory process in a mouse model of asthma.

BACKGROUND: Redox-active iron, a catalyst in the production of hydroxyl radicals via the Fenton reaction, is one of the key participants in ROS-induced tissue injury and general inflammation. According to our recent findings, an excess of tissue iron is involved in several airway-related pathologies such as nasal polyposis and asthma. OBJECTIVE: To examine the anti-inflammatory properties of a newly developed specific iron-chelating complex, Zn/Ga-DFO, in a mouse model of asthma. MATERIALS AND METHODS: Asthma was induced in BALBc mice by ovalbumin, using aluminum hydroxide as an adjuvant. Mice were divided into four groups: (i) control, (ii) asthmatic and sham-treated, (iii) asthmatic treated with Zn/Ga-DFO [intra-peritoneally (i/p) and intra-nasally (i/n)], and (iv) asthmatic treated with Zn/Ga-DFO, i/n only. Lung histology and cytology were examined. Biochemical analysis of pulmonary levels of ferritin and iron-saturated ferritin was conducted. RESULTS: The amount of neutrophils and eosinophils in bronchoalveolar lavage fluid, goblet cell hyperplasia, mucus secretion, and peri-bronchial edema, showed markedly better values in both asthmatic-treated groups compared to the asthmatic non-treated group. The non-treated asthmatic group showed elevated ferritin levels, while in the two treated groups it returned to baseline levels. Interestingly, i/n-treatment demonstrated a more profound effect alone than in a combination with i/p injections. CONCLUSION: In this mouse model of allergic asthma, Zn/Ga-DFO attenuated allergic airway inflammation. The beneficial effects of treatment were in accord with iron overload abatement in asthmatic lungs by Zn/Ga-DFO. The findings in both cellular and tissue levels supported the existence of a significant anti-inflammatory effect of Zn/Ga-DFO.

Degeneration modulates retinal response to transient exogenous oxidative injury.

PURPOSE: Oxidative injury is involved in retinal and macular degeneration. We aim to assess if retinal degeneration associated with genetic defect modulates the retinal threshold for encountering additional oxidative challenges. METHODS: Retinal oxidative injury was induced in degenerating retinas (rd10) and in control mice (WT) by intravitreal injections of paraquat (PQ). Retinal function and structure was evaluated by electroretinogram (ERG) and histology, respectively. Oxidative injury was assessed by immunohistochemistry for 4-Hydroxy-2-nonenal (HNE), and by Thiobarbituric Acid Reactive Substances (TBARS) and protein carbonyl content (PCC) assays. Anti-oxidant mechanism was assessed by quantitative real time PCR (QPCR) for mRNA of antioxidant genes and genes related to iron metabolism, and by catalase activity assay. RESULTS: Three days following PQ injections (1 µl of 0.25, 0.75, and 2 mM) the average ERG amplitudes decreased more in the WT mice compared with the rd10 mice. For example, following 2 mM PQ injection, ERG amplitudes reduced 1.84-fold more in WT compared with rd10 mice (p = 0.02). Injection of 4 mM PQ resulted in retinal destruction. Altered retina morphology associated with PQ was substantially more severe in WT eyes compared with rd10 eyes. Oxidative injury according to HNE staining and TBARS assay increased 1.3-fold and 2.1-fold more, respectively, in WT compared with rd10 mice. At baseline, prior to PQ injection, mRNA levels of antioxidant genes (Superoxide Dismutase1, Glutathione Peroxidase1, Catalase) and of Transferrin measured by quantitative PCR were 2.1-7.8-fold higher in rd10 compared with WT mice (p<0.01 each), and catalase activity was 1.7-fold higher in rd10 (p = 0.0006). CONCLUSIONS: This data suggests that degenerating rd10 retinas encounter a relatively lower degree of damage in response to oxidative injury compared with normal retinas. Constitutive up-regulation of the oxidative defense mechanism in degenerating retinas may confer such relative protection from oxidative injury.

The bitter fate of the sweet heart: impairment of iron homeostasis in diabetic heart leads to failure in myocardial protection by preconditioning.

Cardiovascular dysfunction is a major complication of diabetes. Examining mechanistic aspects underlying the incapacity of the diabetic heart to respond to ischemic preconditioning (IPC), we could show that the alterations in iron homeostasis can explain this phenomenon. Correlating the hemodynamic parameters with levels of ferritin, the main iron storage and detoxifying protein, without and with inhibitors of protein degradation, substantiated this explanation. Diabetic hearts were less sensitive to ischemia-reperfusion stress, as indicated by functional parameters and histology. Mechanistically, since ferritin has been shown to provide cellular protection against insults, including ischemia-reperfusion stress and as the basal ferritin level in diabetic heart was 2-fold higher than in controls, these are in accord with the greater resistance of the diabetic heart to ischemia-reperfusion. Additionally, during ischemia-reperfusion, preceded by IPC, a rapid and extensive loss in ferritin levels, during the prolonged ischemia, in diabetic heart but not in non-diabetic controls, provide additional substantiation to the explanation for loss of respond to IPC. Current research is shedding light on the mechanism behind ferritin degradation as well, suggesting a novel explanation for diabetes-induced loss of cardioprotection.

Are there metallic traces in black extrinsic dental stain?

OBJECTIVE: The detection of ferric ions in samples of black extrinsic dental stain led to the idea that it is comprised of insoluble ferric compounds. The present study examined the chemical composition of black extrinsic dental stain. METHOD AND MATERIALS: Plaque was collected from 17 children with black extrinsic dental stain (study group A) and from 15 children without black extrinsic stain (control group), using sterile graphite curettes; and from 4 children with black extrinsic stain (study group B), using a standard sterile metal curette. Samples were analyzed with a scanning electron microscope (SEM) and subjected to quantitative chemical analysis (energy dispersive spectrometry). RESULTS: Except for calcium and phosphorus levels, no significant differences were found between the chemical composition of black extrinsic dental stain and dental plaque. Metallic ions were not detected in samples collected with a graphite curette (study group A), but were detected in samples collected with a metal curette (study group B). CONCLUSION: Metallic ions do not seem to be the origin of black extrinsic dental stain. Previous reports of the presence of metallic ions are probably due to contamination of the samples by the collection method.

Use of amphoteric rinsing solution for treatment of ocular tissues exposed to nitrogen mustard.

PURPOSE: Ocular exposure to mustard agents may cause severe and prolonged injury to the anterior segment tissues. Effective decontamination of the external eye surface after exposure is of paramount importance. The purpose of the present study was to assess the effectiveness of Diphoterine rinsing solution (DRS) in reducing ocular damage after exposure to nitrogen mustard (NM) and to compare it with normal saline solution. METHODS: One eye of 16 New Zealand albino rabbits was exposed to 2% NM. Immediate thorough irrigation was performed with either 500 ml of DRS (treated group) or with 500 ml of normal saline (control group). The magnitude of ocular injury and response to treatment were assessed by examiners masked to the treatment assignment during 22 days following the exposure. RESULTS: Immediate ocular irrigation with DRS was more effective compared with saline in reducing corneal, iris and anterior chamber injury. In the DRS-treated group, the corneal opacity and corneal neovascularization were less severe, and development of iris atrophy was delayed. Intraocular pressure (mmHg) was better maintained when compared to the control group (day 7 24.3 versus 14.8, p = 0.003; day 12 28 versus 15, p = 0.003; day 22 33.5 versus 21.8, p = 0.014, respectively). Systemic oxidative stress associated with exposure to NM was significantly higher in the saline-treated group than in DRS-treated group (p < 0.011). CONCLUSIONS: The findings of this study indicate the effectiveness of DRS in reducing of NM-induced ocular injuries. Its use should be considered as an immediate treatment modality following exposure to mustard agents to reduce potential ocular injury.

Cardiac protection by preconditioning is generated via an iron-signal created by proteasomal degradation of iron proteins.

Ischemia associated injury of the myocardium is caused by oxidative damage during reperfusion. Myocardial protection by ischemic preconditioning (IPC) was shown to be mediated by a transient 'iron-signal' that leads to the accumulation of apoferritin and sequestration of reactive iron released during the ischemia. Here we identified the source of this 'iron signal' and evaluated its role in the mechanisms of cardiac protection by hypoxic preconditioning. Rat hearts were retrogradely perfused and the effect of proteasomal and lysosomal protease inhibitors on ferritin levels were measured. The iron-signal was abolished, ferritin levels were not increased and cardiac protection was diminished by inhibition of the proteasome prior to IPC. Similarly, double amounts of ferritin and better recovery after ex vivo ischemia-and-reperfusion (I/R) were found in hearts from in vivo hypoxia pre-conditioned animals. IPC followed by normoxic perfusion for 30 min ('delay') prior to I/R caused a reduced ferritin accumulation at the end of the ischemia phase and reduced protection. Full restoration of the IPC-mediated cardiac protection was achieved by employing lysosomal inhibitors during the 'delay'. In conclusion, proteasomal protein degradation of iron-proteins causes the generation of the 'iron-signal' by IPC, ensuing de-novo apoferritin synthesis and thus, sequestering reactive iron. Lysosomal proteases are involved in subsequent ferritin breakdown as revealed by the use of specific pathway inhibitors during the 'delay'. We suggest that proteasomal iron-protein degradation is a stress response causing an expeditious cytosolic iron release thus, altering iron homeostasis to protect the myocardium during I/R, while lysosomal ferritin degradation is part of housekeeping iron homeostasis.

Energy status determines the distinct biochemical and physiological behavior of interfibrillar and sub-sarcolemmal mitochondria.

Reports about the effect of ischemia and reperfusion on specific activities of the respiratory chain are often discrepant. One of the factors that govern this discrepancy is that typical mechanical procedures for mitochondrial isolation yield largely sub-sarcolemmal mitochondria (SSM), while the interfibrillar mitochondria (IFM), which provide most of the energy for the contractile apparatus, are under-represented. Here we investigated the impact of myocardial ischemia and reperfusion on SSM and IFM separately. Thirty-two Wister rats were randomly divided into four groups: control groups, ischemia groups, reperfusion groups and precondition groups. SSM and IFM were isolated from the rats' hearts from all the groups. The mitochondrial membrane potential (Δψ) and swelling were assessed at energized (using either 5mM succinate or 5mM glutamate and 5mM malate (GM) as a substrate) and non-energized conditions, where IFM showed better resistance to change in both conditions. Results showed that IFM have a higher coupling efficiency than SSM when energized by GM, but lower than SSM when energized with succinate. Preconditioning the rats' hearts prior to ischemia or reperfusion preserved the physiological and biochemical functions of both IFM and SSM and are energy dependent. The distinct physiological-biochemical functions of the mitochondrial sub-populations during ischemia and reperfusion depend on the overall energy status of the mitochondrial sub-population.

Rat cardiac mitochondrial sub-populations show distinct features of oxidative phosphorylation during ischemia, reperfusion and ischemic preconditioning.

BACKGROUND: Inter-fibrillar (IFM) and sub-sarcolemmal (SSM) mitochondria are two distinct mitochondrial sub-populations and are expected to behave differently during pathological conditions. This study was undertaken to compare functional oxidative phosphorylation (OXPHOS) in IFM and SSM during ischemia, reperfusion and ischemic preconditioning. METHODS: Langendorff perfused Wistar rat hearts were subjected to 35minutes ischemia, 60minutes reperfusion and ischemic preconditioning (IPC) procedure (3cycles of 2-minutes ischemia followed by 3-minutes reperfusion).Subsequently IFM and SSM were isolated, and mitochondrial electron transport chain (ETC) enzyme activities and respiration were measured immediately. RESULTS: Functional enzyme activities of ETC in IFM and SSM showed prominent differences especially in the proximal part of ETC enzymes during ischemia and reperfusion. SSM favor FADH(2 )while IFM prefer NADH as the main reducing equivalent for electron transport during ischemia and reperfusion. IPC preserved ETC enzyme activities in both IFM and SSM rendering cardio protection. Similarly IPC preserve ADP stimulated respiration with glutamate and malate as substrate in both sub populations, but not in IFM, with succinate as substrate. Apparently, the preconditioning imparts enhanced protection more to SSM than IFM during ischemia and reperfusion and especially to the proximal part of the ETC. CONCLUSION: We propose that mitochondrial dysfunction, one of the major targets of myocardial ischemia reperfusion injury needs to be evaluated by the synergic effect of both IFM and SSM.

Aging is an organ-specific process: changes in homeostasis of iron and redox proteins in the rat.

Organ-specific changes of iron- and redox-related proteins occur with age in the rat. Ferritin, the major iron storage and detoxifying protein, as well as the proteins of the methionine-centered redox cycle (MCRC) were examined in old and young animals, and showed organ-dependent changes. In spleens and livers of aged rats, ferritin (protein) levels were greater than in young ones, and their iron saturation increased, rendering higher ferritin-bound iron (FtBI). Iron saturation of the ferritin molecule in the tongues and sternohyoids of old rats was lower but ferritin level was higher than in young rats, resulting in increased FtBI with age. Ferritin level in the esophagus of older rats was lower than in young rats but its molecular iron content higher thus the total FtBI remained the same. In the larynx, both ferritin and its iron content were the same in young and old animals. MCRC proteins were measured in livers and spleens only. With aging, methionine sulfoxide reductase A and B (MsrA and MsrB) levels in livers and spleens decreased. Thioredoxin1 (Trx) and Trx-reductase1 were elevated in old spleens, but reduced in livers. Aged spleens showed reduced Msr isozyme activity; but in the liver, its activity increased. mRNA changes with age were monitored and found to be organ specific. These organ-specific changes could reflect the different challenges and the selective pathways of each organ and its resultant capacity to cope with aging.

Zinc-desferrioxamine attenuates retinal degeneration in the rd10 mouse model of retinitis pigmentosa.

Iron-associated oxidative injury plays a role in retinal degeneration such as age-related macular degeneration and retinitis pigmentosa. The metallo-complex zinc-desferrioxamine (Zn/DFO) may ameliorate such injury by chelation of labile iron in combination with release of zinc. We explored whether Zn/DFO can affect the course of retinal degeneration in the rd10 mouse model of retinitis pigmentosa. Zn/DFO-treated animals showed significantly higher electroretinographic responses at 3 and 4.5 weeks of age compared with saline-injected controls. Corresponding retinal (photoreceptor) structural rescue was observed by quantitative histological and immunohistochemical techniques. When administered alone, the components of the complex, Zn and DFO, showed a lesser, partial effect. TBARS, a marker of lipid peroxidation, and levels of oxidative DNA damage as quantified by 8-OHdG immunostaining were significantly lower in Zn/DFO-treated retinas compared with saline-injected controls. Reduced levels of retinal ferritin as well as reduced iron content within ferritin molecules were measured in Zn/DFO-treated retinas. The data, taken together, suggest that the protective effects of the Zn/DFO complex are mediated through modulation of iron bioavailability, leading to attenuation of oxidative injury. Reducing iron-associated oxidative stress using complexes such as Zn/DFO may serve as a "common pathway" therapeutic approach to attenuate injury in retinal degeneration.

Effect of para-aminobenzoic acid on the course of retinal degeneration in the rd10 mouse.

PURPOSE: Recent evidence suggests that oxidative injury plays a significant role in the pathogenesis of retinal degenerative diseases. Para-aminobenzoic acid (PABA) is a cyclic amino acid, which may act to decrease lipid peroxidation and oxidative injury. Our aim was to evaluate the efficacy of PABA in attenuating oxidative injury and rate of retinal degeneration in the rd10 mouse. METHODS: PABA (50 mg/kg) was administered intraperitoneally six times per week in 28 rd10 mice from postnatal day 3. Twenty-four littermate control mice were similarly injected with saline. At 3, 4.5, and 6 weeks of age, electrophysiological (full field electroretinogram-ERG), quantitative histological, and immunohistochemical techniques were used to assess the course and extent of retinal degeneration. Degree of lipid peroxidation was determined by the measurement of thiobarbituric acid reactive species (TBARS) and retinal carbonyl content was quantified using the 2,4-dinitrophenylhydrazine method. RESULTS: Dark adapted mixed rod-cone ERG responses at 3 weeks of age were higher in the PABA-treated group as compared to saline control (P < 0.05). By 4.5 weeks, this protective effect was largely abolished and by 6 weeks ERG was unrecordable in both groups. However, at both 3 and 4.5 weeks of age, light-adapted cone ERG amplitudes were better preserved in PABA-treated animals. At 4.5 weeks, thickness of the outer nuclear layer was 28.6% higher in the peripheral retina of PABA-treated mice as compared to controls (P < 0.05). Quantitative immunohistochemistry revealed 2.4-fold higher red/green cone opsin content in the retinas of PABA-treated mice (P < 0.005). At both 3 and 4.5 weeks, levels of TBARS and protein carbonyls were 49%-69% lower in PABA-treated retinas (P < 0.05-0.0005), suggesting less oxidative injury. CONCLUSIONS: PABA treatment may protect retinal function and attenuate the course of retinal degeneration in rd10 mice. Biochemical parameters indicate a lower degree of oxidative injury in PABA-treated retinas. PABA may potentially serve as an addition to antioxidative treatment for retinal and macular degenerations.

Synergistic cytotoxic effect of tetrachlorocatechol and sodium azide in Escherichia coli: toxicity, metabolism, and mechanistic aspects.

Pentachlorophenol (PCP) is used in industrial and domestic applications, including as a biocide and a wood preservative. Metabolism of PCP undergoes oxidative dechlorination, forming tetrachlorocatechol (TCC) and tetrachlorohydroquinone (TCHQ). Both sodium azide (NaN(3)) and TCC appear naturally in soil. None of them are cytotoxic by themselves or facilitate autooxidation. Here, we show that their combination leads to synergistic cytotoxicity (>6 log bacterial killing) to Escherichia coli. The rate of oxygen consumption in a cell-free system showed that NaN(3) increases TCC oxidation by 520-fold. The synergism coefficient to cells was calculated as 96 or greater, and we have shown the formation of a new compound. It is suggested that the intermediate species, o-tetrachlorosemiquinine, and an unknown, nitrogen-centered free radical, both visualized by electron-spin resonance, are harmful species responsible for the synergistic cytotoxicity of TCC/NaN(3), rather than the endproduct formed during the reaction. Desferrioxamine and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide offered nearly complete protection, but through radical scavenging rather than through chelating properties. The mechanism of damage for TCC compared to its analogue, TCHQ, were investigated, and whereas the cellular damage of TCHQ/NaN(3) is through a site-specific mechanism, in the case of TCC/NaN(3) it is through the accumulation of the component(s) in the bacterial cell membrane, eventually leading to dysfunction, as evidenced by electron microscopy.

Alteration in iron metabolism during retinal degeneration in rd10 mouse.

PURPOSE: Altered iron metabolism was implicated in retinal and macular degeneration. This study was designed to further elucidate iron homeostasis during the course of retinal degeneration in mice. METHODS: Retinal mRNA and protein expression of transferrin, transferrin receptor, and ceruloplasmin were evaluated during retinal degeneration in rd10 mice and chemokine receptor 2 (ccr2)-deficient mice. Retinal ferritin protein levels, ferritin-bound iron, and total iron were evaluated in rd10 mice. RESULTS: Transferrin and ceruloplasmin mRNA levels increased between 2- and 12-fold during the course of retinal degeneration in rd10 mice compared with same-age controls (P < 0.01), whereas transferrin receptor mRNA levels increased only at the late stages of degeneration in rd10 mice (2.7-fold; P = 0.005). Transferrin mRNA also increased in retinas of aged ccr2-deficient mice (1.5-fold; P = 0.05). Transferrin and ceruloplasmin protein levels corroborated with mRNA levels changes in rd10 mice albeit at a lower magnitude. Retinal ferritin protein levels increased between 1.5-fold and 2-fold (P < 0.03) in rd10 mice, and ferritin-bound iron levels increased 1.6-fold in 3-week-old rd10 mice (P = 0.03). Three-week-old rd10 mice also had a 1.4-fold increase in total retinal iron level (P = 0.05). CONCLUSIONS: Combined with previous reports, these data suggest that retinal degenerations are associated with altered iron homeostasis regardless of the primary insult. Given the potential of iron to generate oxidative injury, its role as a therapeutic target in retinal and macular degenerations should be evaluated.

Iron, ferritin and proteins of the methionine-centered redox cycle in young and old rat hearts.

Progressive oxidation of cellular components constitutes a major mechanism of the aging process. An emerging paradigm of redox signaling suggests that low level oxidants activate protective pathways resulting in prolonged cell survival. This report centers on the study of cardiac muscle in young and old rats, including (i) the expression of ferritin (Ft) the major iron storage protein, and (ii) the expression of the major proteins of the methionine-centered redox cycle (MCRC), which controls the cellular methionine redox status. Total amounts of Ft (protein) and its mRNA encoding for Ft L-subunit (Ft-L) were higher in the aged hearts, indicating that the iron-binding capacity of myocardial Ft increased with age. Among the proteins of the MCRC, methionine sulfoxide reductases A and B (MsrA, MsrB) and MsrA mRNA were significantly higher in hearts of old rats with a significant decrease in MsrA activity. The observed up-regulation of the expression of Msr and Ft-L could represent a protective response to the increased oxidative stress in the aging myocardium.

Methionine-centered redox cycle in organs of the aero-digestive tract of young and old rats.

It is commonly accepted that aging is associated with a decline in the antioxidant defense of the cell; accordingly, certain redox enzymes are used as markers of biological senescence. To further test and specify this general concept, we studied age-related changes in the enzymes of the methionine-centered redox cycle (MCRC) in four aero-digestive organs of rats. The levels of cytosolic thioredoxin (Trx), thioredoxin reductase (TrxR), and methionine sulfoxide reductase (Msr), all tended to decline with age. The enzymatic activities of MsrA and MsrB were significantly lower in the organs of aged animals. In general, the magnitude of this decline increased in the order: tongue < sternohyoid muscle < larynx < esophagus. The relative stability of MCRC in the old tongues might be part of the well-preserved oxidative metabolism as confirmed by the age-related increase in mitochondrial marker and muscle tissue in these tongues. In total, the results suggest that age-associated oxidative damage is organ-specific and could reflect differences in morphological composition of these tissues, and among them, relative content of striated muscles.