Rate Of Free Radical Production Research Articles

Is the NDUFV2 subunit of the hydrophilic complex I domain a key determinant of animal longevity?

Complex I, a component of the electron transport chain, plays a central functional role in cell bioenergetics and the biology of free radicals. The structural and functional N module of complex I is one of the main sites of the generation of free radicals. The NDUFV2 subunit/N1a cluster is a component of this module. Furthermore, the rate of free radical production is linked to animal longevity. In this review, we explore the hypothesis that NDUFV2 is the only conserved core subunit designed with a regulatory function to ensure correct electron transfer and free radical production, that low gene expression and protein abundance of the NDUFV2 subunit is an evolutionary adaptation needed to achieve a longevity phenotype, and that these features are determinants of the lower free radical generation at the mitochondrial level and a slower rate of aging of long-lived animals.

Free Radical Production, Inflammation and Apoptosis in Patients Treated With Titanium Mandibular Fixations—An Observational Study

Despite high biocompatibility of titanium and its alloys, this metal causes various side effects in the human body. It is believed that titanium biomaterials may induce an innate/adaptive immune response. However, still little is known about changes caused by titanium mandible implants, particularly with regard to bone healing. The latest studies showed disturbances in the antioxidant barrier, increased oxidative/nitrosative stress, as well as mitochondrial abnormalities in the periosteum covering titanium mandible fixations; nevertheless, the impact of titanium implants on free radical production, inflammation, and mandible apoptosis are still unknown. Because severe inflammation and apoptosis are among the main factors responsible for disturbances in osteointegration as well as implant rejection, this study is the first to evaluate pro-oxidant enzymes, cytokines as well as pro- and anti-apoptotic proteins in the periosteum of patients with a broken jaw, treated with titanium miniplates and miniscrews. The study group consisted of 29 patients with double-sided fracture of the mandible body requiring surgical treatment. We found significantly higher activity of NADPH oxidase and xanthine oxidase as well as enhanced rate of free radical production in the periosteum of patients in the study group compared to the control group. The markers of inflammation [interleukin 1 (IL-1), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), transforming growth factor β (TGF-β) and β-glucuronidase (GLU)] as well as apoptosis [Bax, Bax/Bcl-2 ratio, caspase-3 (CAS-3) and nitric oxide (NO)] were significantly elevated in periosteum covering titanium fixations compared to the control group. In the study group, we also demonstrated an increased content of titanium on the periosteum surface, which positively correlated with CAS-3 activity. The study led us to the conclusion that titanium mandible implants increase the production of pro-inflammatory cytokines, and enhance free radical generation in the periosteum covering titanium miniplates and miniscrews. Additionally, exposure to Ti6Al4V titanium alloy induces apoptosis in the mandible periosteum. However, no clinical signs of the said phenomena have been observed.

Chlorinated metronidazole as a promising alternative for treating trichomoniasis.

Trichomoniasis is the most common non-viral, sexually transmitted infection affecting humans worldwide. The main treatment for trichomoniasis is metronidazole (MTZ). However, adverse effects and reports of resistance have stimulated the development of therapeutic alternatives. The ease of manipulation of the side chains of MTZ coupled with its safety makes this molecule attractive for the development of new drugs. In this context, we evaluated the activity of the chlorinated MTZ derivative, MTZ-Cl, on sensitive and resistant strains of Trichomonas vaginalis. MTZ-Cl presented a remarkable activity against both sensitive and resistant strains. In vitro and in vivo toxicity assays indicated that the new molecule is safe for future clinical trials. Furthermore, we noticed different rates of free radical production between the sensitive and resistant strains. MTZ-Cl induced a higher release of nitric oxide (NO, ~ 9000a.u.) by both sensitive and resistant strains. However, the sensitive strain produced a greater amount of H2O2 (~ 1,800,000a.u.) and superoxide radicals (~ 350,000a.u.) in the presence of MTZ. In the resistant strain, production of these radicals was more prominent when MTZ-Cl was used. Collectively, these results suggest that NO is an important molecule in the trichomonacidal activity against resistant and sensitive strains, suggesting an alternative pathway for MTZ-Cl activation. We highlight the high trichomonacidal potential of MTZ-Cl, improving the effectiveness of treatment and reducing side effects. In addition, MTZ-Cl is derived from a well-established drug on the world market that presents low toxicity to human cells, suggesting its safety to proceed with future clinical trials.

Effects of Grape Wine and Apple Cider Vinegar on Oxidative and Antioxidative Status in High Cholesterol-Fed Rats

Background: Oxidative stress is the result of an imbalance between the rates of free radical production and elimination via endogenous antioxidant mechanisms such as antioxidant enzymes; glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT). Antioxidants widely available in fruits, vegetables, seeds have been possessed a broad spectrum of biological, pharmacological and therapeutic properties against oxidative stress. Consumption of fruits and vegetables are essentials much as their products such as fruit juices, wines and vinegars, which contain significant amount of polyphenolic compounds. Vinegar is produced mainly from different varieties of wine by two fermentation process, ethanol and acetic acid fermentations. Followed by wine production there are mainly two vinegar production methods. One is surface also known as traditional method. The second method is submerging technique involving submerged culture where the oxygenation has been greatly improved (industrial method).Objective: The aim of the study is to determine the effects of grape and apple cider vinegar consumption against oxidative stress in high cholesterol-fed rats.Methods: Fifty-four male, adult Wistar albino rats were included in the study. Rats were divided into six groups of nine. 1 mL of 2.5% cholesterol (at 5pm) and 1 mL of different vinegar samples (at 9 am) were administered daily for 7 weeks by oral gavage. Control-diet group (CNT) received 1mL of normal saline solution concurrently with the experiment groups. Rats were sacrificed at the end of the experiment and blood samples were collected. The erythrocyte samples were washed three times in normal saline (0.9%, v/w) and then hemolyzed with 2mL of cold bidistillated water. CAT activity was measured following the method of Aebi. MDA was determined by the double heating method of Draper and Hadley. GSH-Px activity was measured according to the method of Paglia and Valentine [19]. SOD activity was analyzed according to the method of Woolliams et al.[20] Both were analyzed in Beckmann Coulter AU 5800 autoanalyzer by using RANDOX kits (Randox Laboratories Ltd. Ardmore, Crumlin, UK). Vinegars were obtained after the grape and apple vinegar fermentations using surface culture method and industrial submerge methods. Grape and apple juices were immediately inoculated with Saccharomyces cerevisiae (0.02%) for ethanol fermentation for 30 day at 25°C. After the completion of the ethanol fermentation, acetic acid fermentation of wines was initiated with the addition of two-year aged vinegar (1:3 ratio) using surface technique at 25°C and continued for 60 days at 25°C.Vinegars produced by the industrial submerge method for 24 hours at 25°Cwere transported to theDepartment of Food Engineering laboratories from the Carl Kuhne Vinegar Plant located in Afyonkarahisar, Turkey. Total antioxidant activity of vinegar samples were measured by Oxygen Radical Absorbance Capacity (ORAC) and 2,2’-azinobis (3-ethlybenzthiazoline)-6-sulfonic acid (ABTS) methods.Results: Levels of CAT, GSH-Px, SOD in CHCNT group were significantly decreased while MDA levels were significantly increased when compared to CNT group. Levels of MDA which is the end-product of lipid peroxidation was significantly decreased in the apple cider vinegar administered groups (TAV and IAV) when compared to the CHCNT (P<0.05). MDA levels of grape wine vinegar administered groups were decreased (TGV, IGV), however the difference was not significant. GSH-Px levels were significantly increased in both TGV and TAV groups, which were fed with the vinegars produced by traditional surface methods (P=0.03, P=0.001 respectively) as compared to the CHCNT. GSH-Px levels of rats fed with vinegars produced with industrial submerge methods (IGV, IAV), showed no significant difference when compared to CHCNT group. SOD levels of TGV, IGV, TAV, IAV were significantly increased as compared to CHCNT group (p<0.05). TEAC and ORAC values of vinegar samples (TGV and TAV) produced with surface methods were higher than other samples. ORAC and TEAC values of TAV sample was 5.89 µmol trolox/ml and 5.5 mM, respectively.Conclusions: Present research showed that high cholesterol diet increased lipid peroxidation and consumed the antioxidant enzymes. Although the degree of the effect of vinegars on antioxidant enzyme activity differs, the use of vinegar especially the ones produced by surface culture methods have seem to have favorable effect in vivo. These findings are in concordance with the ORAC and TEAC values of vinegars.Keywords: Oxidative stress, grape vinegar, apple cider vinegar, glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT)

Chronic α-Tocopherol Increases Central Monoamines Synthesis and Improves Cognitive and Motor Abilities in Old Rats.

Limiting enzymes in the synthesis of brain monoamines seems to be susceptible to oxidative damage, one of the most important factors in aging. It has been suggested that the use of anti-oxidants can reduce the rate of free radical production related with aging and the associated damage. Therefore, this study aims to analyze the effects of the chronic treatments with the anti-oxidant α-tocopherol (vitamin E) on central monoamines (high-performance liquid chromatography [HPLC] analysis) mediating cognitive functions, as well as on the evaluation of memory and motor abilities in old rats measured by radial maze, Barnes maze, novel object recognition test, and rotarod test. Results show that α-tocopherol significantly increased in a dose- and/or time-dependent manner the synthesis rate and the levels of monoaminergic neurotransmitters (serotonin, dopamine, and noradrenaline) in the hippocampus and striatum, brain regions involved in memory processing and motor coordination. These positive neurochemical effects, largely due to an increased activity of the limiting enzymes in monoamines synthesis, tryptophan hydroxylase and tyrosine hydroxylase, were accompanied by an improvement in cognitive and motor abilities in old rats. Altogether these findings suggest that α-tocopherol exhibits neuroprotective actions in old rats; thus, diets with α-tocopherol might represent a promising strategy to mitigate or delay the cognitive and motor decline associate with aging and related-diseases.

NLRP3 Inflammasome is activated in the kidneys of Metabolic Syndrome animals

Inflammasomes are oligomeric protein complexes formed in response to Danger‐associated molecular patterns (DAMPS) such as uric acid crystals and asbestos and have been shown to contribute to the pathogenesis of metabolic syndrome (MetS). Since uric acid is increased in chronic kidney disease, we hypothesized that increase uric acid might induce NLRP3 inflammasomes and exacerbate MetS via disrupting the redox balance and causing structural and functional impairment of the kidneys. Male Lean zucker controls (LZR) and Obese (OZR) underwent acute renal clearance and hemodynamic study. Compared to the OZR rats, the LZR exhibited higher glomerular filtration rate, higher renal blood flow and lower renal vascular resistance. Renal histopathology results revealed glomerulosclerosis, interstitial nephritis and proteinuria, and Real time RT PCR showed an increased gene expression of kidney injury markers and inflammasomes in OZR rats. Free radical production rates measured by electron paramagnetic spectroscopy showed a reduced total reactive oxygen species, superoxide and peroxynitrite levels in LZR animals. The gene and protein expression levels of IL‐1β and IL‐18 were higher in OZR rats. The plasma uric acid levels were higher in OZR rats. Our results indicate that inflammasomes are activated in the kidneys of MetS, thereby contributing to increased oxidative stress and renal damage. Research supported by LSU.

Blueberry treatment improves renal function and reduces oxidative stress in Metabolic Syndrome animals – Role of Toll‐like receptor 4 (TLR4)

Metabolic syndrome (MetS) is characterized by a cluster of health factors that indicate a higher risk for cardiac disease, renal disabilities and diabetes. Although inflammation and oxidative stress in renal dysfunction has been well studied, the mechanism by which natural antioxidants protect against MetS is not clear. Blueberries (BB) are among the richest source of antioxidants known to have anti‐inflammatory and antioxidant properties. We hypothesize that BB protect against MetS via inhibition of TLR4, which is an upstream receptor in several inflammatory pathways. Male zucker rats – Lean controls (LZR) and Obese (OZR) were both fed with a BB‐enriched diet or a control diet for 15 weeks. Compared to controls, the rats fed with BB exhibited higher glucose sensitivity, lower blood pressure and improved renal function. Free radical production rates measured by electron paramagnetic spectroscopy (EPR) showed a decreased total reactive oxygen species, superoxide and peroxynitrite levels in BB treated rats. Reduction in renal pathology and improvement in antioxidant levels in the BB‐fed rat kidneys were consistent with the EPR results. BB treated animals also expressed lower gene and protein levels of TLR4 and proinflammatory cytokines. In conclusion, TLR4 inhibition is a mechanism by which blueberries protect against oxidative stress and renal damage, and prevent the onset of MetS. Research supported by NIH.

Bioenergetic Consequences of PINK1 Mutations in Parkinson Disease

BackgroundMutations of the gene for PTEN-induced kinase 1 (PINK1) are a cause of familial Parkinson's disease (PD). PINK1 protein has been localised to mitochondria and PINK1 gene knockout models exhibit abnormal mitochondrial function. The purpose of this study was to determine whether cells derived from PD patients with a range of PINK1 mutations demonstrate similar defects of mitochondrial function, whether the nature and severity of the abnormalities vary between mutations and correlate with clinical features.MethodologyWe investigated mitochondrial bioenergetics in live fibroblasts from PINK1 mutation patients using single cell techniques. We found that fibroblasts from PINK1 mutation patients had significant defects of bioenergetics including reduced mitochondrial membrane potential, altered redox state, a respiratory deficiency that was determined by substrate availability, and enhanced sensitivity to calcium stimulation and associated mitochondrial permeability pore opening. There was an increase in the basal rate of free radical production in the mutant cells. The pattern and severity of abnormality varied between different mutations, and the less severe defects in these cells were associated with later age of onset of PD.ConclusionsThe results provide insight into the molecular pathology of PINK1 mutations in PD and also confirm the critical role of substrate availability in determining the biochemical phenotype – thereby offering the potential for novel therapeutic strategies to circumvent these abnormalities.

Chronic exercise preserves renal structure and hemodynamics in spontaneously hypertensive rats.

Exercise training (ExT) is a recommended adjunct to many pharmaceutical antihypertensive therapies. The effects of chronic ExT on the development of hypertension-induced renal injury remain unknown. We examined whether ExT would preserve renal hemodynamics and structure in the spontaneously hypertensive rat (SHR), and whether these effects were mediated by improved redox status and decreased inflammation. Normotensive WKY rats and SHR underwent moderate-intensity ExT for 16 weeks. One group of SHR animals was treated with hydralazine to investigate the pressure-dependent/independent effects of ExT. Acute renal clearance experiments were performed prior to sacrifice. Tissue free radical production rates were measured by electron paramagnetic resonance; gene and protein expression were measured by real time RT-PCR and Western blot or immunofluorescence, respectively. Plasma angiotensin II levels and kidney antioxidants were assessed. Training efficacy was assessed by citrate synthase activity assay in hind-limb muscle. ExT delayed hypertension, prevented oxidative stress and inflammation, preserved antioxidant status, prevented an increase in circulating AngII levels, and preserved renal hemodynamics and structure in SHR. In addition, exercise-induced effects, at least, in part, were found to be pressure-independent. This study is the first to provide mechanistic evidence for the renoprotective benefits of ExT in a model of hypertension. Our results demonstrate that initiation of ExT in susceptible patients can delay the development of hypertension and provide renoprotection at the functional and ultrastructural level. Chronic ExT preserves renal hemodynamics and structure in SHR; these effects are partially mediated by improved redox status and decreased inflammation.

Evolution of Senescence in Nature: Physiological Evolution in Populations of Garter Snake with Divergent Life Histories

Evolutionary theories of aging are linked to life-history theory in that age-specific schedules of reproduction and survival determine the trajectory of age-specific mutation/selection balances across the life span and thus the rate of senescence. This is predicted to manifest at the organismal level in the evolution of energy allocation strategies of investing in somatic maintenance and robust stress responses in less hazardous environments in exchange for energy spent on growth and reproduction. Here we report experiments from long-studied populations of western terrestrial garter snakes (Thamnophis elegans) that reside in low and high extrinsic mortality environments, with evolved long and short life spans, respectively. Laboratory common-environment colonies of these two ecotypes were tested for a suite of physiological traits after control and stressed gestations. In offspring derived from control and corticosterone-treated dams, we measured resting metabolism; mitochondrial oxygen consumption, ATP and free radical production rates; and erythrocyte DNA damage and repair ability. We evaluated whether these aging biomarkers mirrored the evolution of life span and whether they were sensitive to stress. Neonates from the long-lived ecotype (1) were smaller, (2) consumed equal amounts of oxygen when corrected for body mass, (3) had DNA that damaged more readily but repaired more efficiently, and (4) had more efficient mitochondria and more efficient cellular antioxidant defenses than short-lived snakes. Many ecotype differences were enhanced in offspring derived from stress-treated dams, which supports the conclusion that nongenetic maternal effects may further impact the cellular stress defenses of offspring. Our findings reveal that physiological evolution underpins reptilian life histories and sheds light on the connectedness between stress response and aging pathways in wild-dwelling organisms.

Blood lead: Its effect on trace element levels and iron structure in hemoglobin

Lead is a ubiquitous environmental pollutant that induce a broad range of physiological and biochemical dysfunctions. The purpose of this study was to investigate its effects on trace elements and the iron structure in hemoglobin. Blood samples were collected from rats that had been exposed to lead. The concentration of trace elements in whole blood and blood plasma was determined by ICP-MS and the results indicate that lead exists mainly in the red blood cells and only about 1–3% in the blood plasma. Following lead exposure, the concentrations of zinc and iron in blood decrease, as does the hemoglobin level. This indicates that the heme biosynthetic pathway is inhibited by lead toxicity and that lead poisoning-associated anemia occurs. The selenium concentration also decreases after lead exposure, which may lead to an increased rate of free radical production. The effect of lead in the blood on iron structure in hemoglobin was determined by EXAFS. After lead exposure, the Fe–O bond length increases by about 0.07 Å and the Fe–Np bond length slightly increases, but the Fe–N ε bond length remains unchanged. This indicates that the blood content of Hb increases, but that the content of HbO 2 decreases.

Reperfusion injury and inflammatory responses following acute lower limb revascularization surgery

After revascularization of an acute arterial occlusion the development of a serious ischaemic-reperfusion injury is a menacing challenge and a hard task in peripheral vascular surgery. A whale of evidences point to oxidative stress, as an important trigger, in the complex chain of events leading to reperfusion injury. In the present study authors aimed to examine oxidative stress parameters, antioxidant-prooxidant state and leukocyte adhesion molecules (CD11a and CD18) expression following acute revascularization surgery of lower limb.10 patients were examined in the prospective randomized study. Peripheral blood sample was collected in ischaemic period, and after reperfusion in the 2nd and 24th hours, and on 7th day. Superoxide-dismutase activity, reduced glutathion concentration and leukocytes free radical production were measured. The degree of lipidperoxidation was marked with the quantity of malondialdehyde. The expressions of adhesion molecules were measured with flowcytometry.The speed and rate of free radical production significantly increased in the early reperfusion (p<0.05). The level of antioxidant enzymes decreased after revascularization. The CD11a and CD18 expression of the granulocytes significantly (p<0.05) decreased right after the revascularization, but with a gradual elevation until the 7th day they exceed the ischaemic value. Our results showed a time specific turnover of the sensitive antioxidant-prooxidant balance after revascularization operation.

Avian senescence: underlying mechanisms

Candidate mechanisms for physiological aging include free radical production and resulting oxidative damage, progressive erosion of telomeres and cellular senescence, age-dependent trade-offs in hormone signaling pathways, and immunosenescence, leading to an increased risk of infection, autoimmune disease, and cancer. These mechanisms are inter-related, not mutually exclusive, and probably all contribute to the aging phenotype. To date, most studies on mechanisms of aging are based on cell culture or lab animals, but interest in comparative studies is growing rapidly. Compared to mammals, birds have long life spans for their body sizes. Birds also appear to have lower rates of free radical production and oxidative damage than mammals, despite higher levels of oxidative metabolism. High levels of the antioxidant, uric acid, in birds may help protect against oxidative damage. Cultured bird cells are more resistant to oxidative damage than mammal cells, and membrane phospholipids of birds are less susceptible to peroxidation than those of mammals of the same size, but show a similar susceptibility as those of mammals with the same life span. In birds, telomeres shorten with age, and the rate of shortening is proportional to life span. Telomerase has a higher activity in long-lived than in short-lived species. Within a species, short telomeres correlate with reduced survival. Birds have higher plasma glucose than mammals, but lower levels of protein glycation, which contributes to aging damage. Immunosenescence is linked to both oxidative damage and telomere shortening. Patterns of cellular and humoral immunosenescence differ among species in birds. The rate of decline in cell-mediated immune function is inversely correlated with life span. Comparative studies on mechanisms underlying senescence in birds will continue to provide us with valuable information on how aging mechanisms have evolved.

From benefit to damage. Glutamate and advanced glycation end products in Alzheimer brain

The glutamatergic system is the most widespread neurotransmitter system in the mammalian brain. It is connected to the acetylcholinergic neurotransmitter system to form the glutamatergic/aspartatergic-acetylcholinergic circuit, which is the morphobiochemical basis of learning, memory and cognition assisted by the glutamatergic N-methyl-D-aspartate receptor, which mediates long-term potentiation as the fundamental molecular mechanisms of these mental capacities. Glutamate and acetylcholine as ligands of the two neurotransmitter systems are products of the neuronal glucose metabolism as holds true also for advanced glycation end products (AGEs), which are markers of damaged and/or aged proteins. During normal aging, both the neurotransmitters glutamate and acetylcholine undergo strong functional variations. Their synthesis was found to be reduced as a common feature. In contrast, basal release of acetylcholine and receptor number decrease, whereas basal release of glutamate and receptor number increase. AGEs increase during aging obviously preferentially in glutamatergic pyramidal neurons in cerebral cortical layers prone to neurodegeneration. In sporadic Alzheimer disease (SAD), glutamate concentration was shown to fall since it may serve as a substitute for lacking glucose in the beginning of the disease. In contrast, glutamate receptor density was found to be much less involved indicating an excessive activation of the glutamatergic neurotransmitter system particularly via the NMDA receptor, mediating endogenous excitotoxicity. The morphological hallmarks of SAD neuritic plaques and neurofibrillary tangles have been demonstrated to crosslink with AGEs causing an increased rate of free radical production. First data from animal studies and investigations on human beings may indicate that the NMDA receptor antagonist memantine may have beneficial effects on the course of SAD and its clinical symptoms.

Changes in the level of antioxidants in the blood from mice infected with Trichinella spiralis.

The biological reaction caused by oxygen-derived free radicals at the molecular and cellular levels involves many different biochemical components which can be directly damaged by oxidizing radicals. As such a reaction may lead to pathological processes, defence mechanisms have evolved to limit the rate of free radical production. These mechanisms employ low-molecular-weight non-enzymatic antioxidants and antioxidant enzymes which are inducible by oxidant stress. In this study, the activity of two antioxidant enzymes, superoxide dismutase (EC 1.15.1.1) and glutathione peroxidase (EC 1.11.1.9), and the level of non-enzymatic antioxidants (total antioxidant status) in the blood from mice infected with Trichinella spiralis was examined. We observed a statistically significant, up to above twofold increase (relative to the control value in uninfected mice) in the level of both enzymes as well as in the total antioxidant status. An intensification of antioxidant processes during trichinellosis could be related to the presence of T. spiralis larvae, which may induce phagocytes to generate free radicals. Our research shows that the maximum growth in antioxidant activity in the blood appears during the period of the greatest muscle damage caused by T. spiralis infection at 3-7 weeks post-infection.

Effect of time of restriction on the decrease in mitochondrial H2O2 production and oxidative DNA damage in the heart of food-restricted rats.

In the present study, the question if medium-term (4 months) caloric restriction (40%) decreases mitochondrial H2O2 production and oxidative DNA damage was investigated. Caloric restriction (CR) is the only experimental manipulation that increases maximum life span. Previous long-term CR studies have showed that CR decreases the mitochondrial rate of free radical production in diverse tissues and species. Those studies agree with the idea that the superior longevity of the restricted animals can be partly due to their lower mitochondrial rate of free radical generation. However, caloric restriction effects strongly depend on implementation time. Previous studies have shown that the decrease induced by CR on oxygen radical generation and oxidative damage to mitochondrial DNA occurs after 1 year but not after 6 weeks of restriction. In the present investigation, mitochondrial H2O2 production did not change in medium-term (4 months) caloric restricted animals, and, in agreement with that, no differences were found in either mitochondrial or nuclear oxidative DNA damage between restricted and ad libitum-fed animals. These results confirm the importance of the time of CR implementation, and show that time longer than 4 months is needed to decrease the mitochondrial rate of free radical generation and the oxidative damage to mtDNA in the rat heart.

Iron toxicity and chelation therapy.

Iron is an essential mineral for normal cellular physiology, but an excess can result in cell injury. Iron in low-molecular-weight forms may play a catalytic role in the initiation of free radical reactions. The resulting oxyradicals have the potential to damage cellular lipids, nucleic acids, proteins, and carbohydrates; the result is wide-ranging impairment in cellular function and integrity. The rate of free radical production must overwhelm the cytoprotective defenses of cells before injury occurs. There is substantial evidence that iron overload in experimental animals can result in oxidative damage to lipids in vivo, once the concentration of iron exceeds a threshold level. In the liver, this lipid peroxidation is associated with impairment of membrane-dependent functions of mitochondria and lysosomes. Iron overload impairs hepatic mitochondrial respiration primarily through a decrease in cytochrome C oxidase activity, and hepatocellular calcium homeostasis may be compromised through damage to mitochondrial and microsomal calcium sequestration. DNA has also been reported to be a target of iron-induced damage, and this may have consequences in regard to malignant transformation. Mitochondrial respiratory enzymes and plasma membrane enzymes such as sodium-potassium-adenosine triphosphatase (Na(+) + K(+)-ATPase) may be key targets of damage by non-transferrin-bound iron in cardiac myocytes. Levels of some antioxidants are decreased during iron overload, a finding suggestive of ongoing oxidative stress. Reduced cellular levels of ATP, lysosomal fragility, impaired cellular calcium homeostasis, and damage to DNA all may contribute to cellular injury in iron overload. Evidence is accumulating that free-radical production is increased in patients with iron overload. Iron-loaded patients have elevated plasma levels of thiobarbituric acid reactants and increased hepatic levels of aldehyde-protein adducts, indicating lipid peroxidation. Hepatic DNA of iron-loaded patients shows evidence of damage, including mutations of the tumor suppressor gene p53. Although phlebotomy therapy is effective in removing excess iron in hereditary hemochromatosis, chelation therapy is required in the treatment of many patients who have combined secondary and transfusional iron overload due to disorders in erythropoiesis. In patients with beta-thalassemia who undergo regular transfusions, deferoxamine treatment has been shown to be effective in preventing iron-induced tissue injury and in prolonging life expectancy. The use of the oral chelator deferiprone remains controversial, and work is continuing on the development of new orally effective iron chelators.

Impaired iron homeostasis in Parkinson's disease.

Despite physiological systems designed to achieve iron homeostasis, increased concentrations of brain iron have been demonstrated in a range of neurodegenerative diseases. These including the parkinsonian syndromes, the trinucleotide repeat disorders and the dementia syndromes. The increased brain iron is confined to those brain regions most affected by the degeneration characteristic of the particular disorder and is suggested to stimulate cell damage via oxidative mechanisms. Changes in central iron homeostasis have been most closely investigated in PD, as this disorder is well characterised both clinically and pathologically. PD is associated with a significant increase in iron in the degenerating substantia nigra (SN) and is measureable in living PD patients and in post-mortem brain. This increase, however, occurs only in the advanced stages of the disease, suggesting that this phenonoma may be a secondary, rather than a primary initiating event, a hypothesis also supported by evidence from animal experiments. The source of the increased iron is unknown but a variety of changes in iron homeostasis have been identified in PD, both in the brain and in the periphery. The possibility that an increased amount of iron may be transported into the SN is supported by data demonstrating that one form of the iron-binding glycoprotein transferrin family, lactotransferrin, is increased in surviving neurons in the SN in the PD brain and that this change is associated with increased numbers of lactotransferrin receptors on neurons and microvessels in the parkinsonian SN. These changes could represent one mechanism by which iron might concentrate within the PD SN. Alternatively, the measured increased in iron might result from a redistribution of ferritin iron stores. Ferritin is located in glial cells while the degenerating neurons do not stain positive for ferritin. As free radicals are highly reactive, it is unlikely that glial-derived free radicals diffuse across the intracellular space in sufficent quantities to damage neuronal constituents. If intracellular iron release contributes to neuronal damage it seems more probable that an intraneuronal iron source is responsible for oxidant-mediated damage. Such a iron source is neuromelanin (NM), a dark-coloured pigment found in the dopaminergic neurons of the human SN. In the normal brain, NM has the ability to bind a variety of metals, including iron, and increased NM-bound iron is reported in the parkinsonian SN. The consequences of these phenomena for the cell have not yet been clarified. In the absence of significant quantities of iron NM can act as an antioxidant, in that it can interact with and inactivate free radicals. On the other hand, in the presence of iron NM appears to act as a proxidant, increasing the rate of free radical production and thus the oxidative load within the vulnerable neurons. Given that increased iron is only apparent in the advanced stages of the disease it is unlikely that NM is of importance for the primary aetiology of PD. A localised increase in tissue iron and its interaction with NM may be, however, important as a secondary mechanism by increasing the oxidative load on the cell, thereby driving neurodegeneration.

36] Gas chromatography-mass spectrometry analysis of DNA: Optimization of protocols for isolation and analysis of DNA from human blood

This chapter shows that 5 ml of whole blood, or packed cells from this volume, is required to extract 100μg DNA, which allows easy analysis of base damage by GC/MS, and storing blood at -20 ° for up to 1 year does not affect DNA damage. Although the DNA extraction protocol followed in this study requires removal of RNA to prevent artifacts in DNA base damage determination, showing a lack of effect with all the other conditions tested, it is not necessary that every DNA extraction procedure follows this pattern. The optimal protocol may depend on sources of reagents and the general level of contamination with prooxidants (e.g., transition metal ions in the water and reagents) in the laboratory concerned. It has subsequently applied this protocol to the investigation of factors affecting oxidative DNA damage in the human body. It is found that steady-state levels of oxidative DNA damage varied widely among healthy individuals. This may be related to a number of endogenous and exogenous factors, including genetic predisposition, exposure to environmental toxins, and antioxidant intake. Levels of DNA damage and repair is also influenced by endogenous rates of free radical production and level of antioxidant defenses, as well as the activity of repair enzymes and the ability to respond positively to increased levels of oxidative stress, including the absorption, processing, and transport of dietary antioxidants to the site of oxidative stress. Collectively, such factors contribute to the overall levels of oxidative stress in the human body and thus DNA damage and, in the long term, perhaps susceptibility to cancer.

Free radical activity, antioxidant enzyme, and glutathione changes with muscle stretch injury in rabbits.

The present study investigated changes in rate of free radical production, antioxidant enzyme activity, and glutathione status immediately after and 24 h after acute muscle stretch injury in 18 male New Zealand White rabbits. There was no change in free radical production in injured muscles, compared with noninjured controls, immediately after injury (time 0; P = 0.782). However, at 24 h postinjury, there was a 25% increase in free radical production in the injured muscles. Overall, there was an interaction (time and treatment) effect (P = 0.005) for free radical production. Antioxidant enzyme activity demonstrated a treatment (injured vs. control) and interaction effect for both glutathione peroxidase (P = 0.015) and glutathione reductase (P = 0.041). There was no evidence of lipid peroxidation damage, as measured by muscle malondialdehyde content. An interaction effect occurred for both reduced glutathione (P = 0.008) and total glutathione (P = 0.015). Morphological analysis (hematoxylin and eosin staining) showed significant polymorphonuclear cell infiltration of the damaged region at 24 h postinjury. We conclude that acute mechanical muscle stretch injury results in increased free radical production within 24 h after injury. Antioxidant enzyme and glutathione systems also appear to be affected during this early postinjury period.