Mechanical strain increases protein tyrosine phosphorylation in airway smooth muscle cells

The 'comet' assay is being increasingly employed for evaluating DNA damage in biological systems. Using this technique, we examined DNA damage in whole in density-separated trout erythrocytes. Results clearly show that all the three considered parameters (tail length, tail intensity and tail moment) increased with the density of the fractions, possibly reflecting different degrees of DNA damage. Probably, this behaviour is due to different periods of exposure of the density fractions to the hazard of active oxygen radicals; older cells have been exposed to oxidative stress for a longer time.     

Artificial background and induced levels of oxidative base damage in DNA from human cells

The pre-mutagenic oxidative DNA base damage of 8-hydroxy-guanine is present in DNA isolated from cells and the amount present increases with exposure of cells to oxidative stress. The oxidative DNA base damage may be present before isolation of DNA or it may be produced during isolation and processing of DNA. We have found that the amount of oxidative base damage measured in DNA can be reduced to a stable lower level by adding increasing concentrations of the antioxidants desferrioxamine, histidine and reduced glutathione immediately before cell lysis. Inclusion of these antioxidants after cell lysis did not affect the level of DNA damage. Oxidative DNA base damage produced by ultraviolet A irradiation of human cells was also reduced by adding antioxidants after irradiation and before cell lysis. Thus, unidentified oxidants induced by ultraviolet A irradiation may damage DNA significantly during extractions of DNA from cells subsequent to ultraviolet A irradiation.     

Oxidative stress in critical care: is antioxidant supplementation beneficial?

Reactive oxygen species (ROS) are constantly produced in human beings under normal circumstances. Antioxidant systems help defend the body against ROS but may be overwhelmed during periods of oxidative stress, which can cause lipid peroxidation, damage to DNA, and cell death. Critical illness, such as sepsis or adult respiratory distress syndrome, can drastically increase the production of ROS and lead to oxidative stress. Sources of oxidative stress during critical illness include activation of the phagocytic cells of the immune system (the respiratory burst), the production of nitric oxide by the vascular endothelium, the release of iron and copper ions and metalloproteins, and the vascular damage caused by ischemia reperfusion. Only indirect measurements of ROS are available, but the presence of oxidative stress in critical illness is supported by clinical studies. In general, serum antioxidant vitamin concentrations seem to decrease and measures of oxidative stress seem to increase in critically ill populations. Oxidative stress has been associated with sepsis, shock, a need for mechanical ventilation, organ dysfunction, acute respiratory distress syndrome, disseminated intravascular coagulation, surgery, and the presence of an acute-phase response. In addition, higher levels of oxidative stress seem to occur in patients with more notable injuries. Dietary supplementation with antioxidant vitamins seems to be the logical answer to decreasing serum antioxidant concentrations, but antioxidants may have adverse effects. The benefit of supplementing antioxidants in critically ill populations has not been shown and requires further study.     

Oxidative damage does not alter membrane phospholipid asymmetry in human erythrocytes

Oxidant-induced damage has been proposed to be the underlying mechanism for loss of membrane phospholipid asymmetry in the erythrocyte membrane. In sickle cell disease, thalassemia, and diabetes as well as in senescent erythrocytes, an apparent correlation between oxidative damage and loss of phosphatidylserine asymmetry has been reported. In the present study, erythrocytes were subjected to various levels of oxidative stress and/or sulfhydryl modifying agents. The transmembrane location of phosphatidylserine (PS) was assessed by FITC-conjugated annexin V labeling and the PS-dependent prothrombinase assay. Transbilayer movement of spin-labeled PS was used to determine aminophospholipid translocase activity. Our data show that cells did not expose PS as the result of oxidative stress induced by phenylhydrazine, hydrogen peroxide, tert-butyl hydroperoxide, cumene hydroperoxide, or sulfhydryl modification by N-ethylmaleimide (NEM) and diamide, even under conditions that led to severe cellular damage and impairment of aminophospholipid translocase activity. In contrast, the increase of intracellular calcium induced by treatment with calcium and ionophore A23187 leads to a rapid scrambling of the lipid bilayer and the exposure of PS, which can be exacerbated by the inhibition of aminophospholipid translocase activity. Oxidation of the cells with hydrogen peroxide or phenylhydrazine did not affect A23187-induced uptake of calcium, but partly inhibited calcium-induced membrane scrambling. In conclusion, oxidative damage of erythrocytes does not induce exposure of phosphatidylserine on the membrane surface, but can interfere with both aminophospholipid translocase activity and calcium-induced randomization of membrane phospholipids.     

Renal clearance, tissue distribution, and CA-125 responses in a phase I trial of suramin

Exposure of human spermatozoa to nicotinamide adenine dinucleotide phosphate (NADPH) resulted in the dose dependent generation of reactive oxygen species (ROS) which, at a critical level of intensity, induced lipid peroxidation, DNA damage and a dramatic decline of sperm motility. This system was then used as a model for screening the ability of different antioxidants to combat oxidative stress created through the excessive intracellular generation of toxic oxygen products of metabolism. A variety of antioxidants that has previously been shown to be protective against extracellularly derived oxidants (e.g. superoxide dismutase, catalase, vitamin E, hypotaurine) were ineffective in this system. Albumin, however, could provide complete protection against NADPH induced oxidative stress via mechanisms that did not involve the suppression of the lipid peroxidation cascade but rather the inactivation of lipid peroxides generated during this process. Albumin did not protect against DNA damage induced by NADPH but was extremely effective at preventing DNA fragmentation arising from the suppression of glutathione peroxidase activity with mercaptosuccinate. These studies emphasize that the design of clinically effective antioxidant treatments will depend, critically, upon the source of the oxidative stress. For cases involving excessive intracellular ROS generation, albumin appears to be an important means of neutralizing lipid peroxide-mediated damage to the sperm plasma membrane and DNA.     

A study and meta-analysis of lay attributions of cures for overcoming specific psychological problems

1. Lipoic acid is an example of an existing drug whose therapeutic effect has been related to its antioxidant activity. 2. Antioxidant activity is a relative concept: it depends on the kind of oxidative stress and the kind of oxidizable substrate (e.g., DNA, lipid, protein). 3. In vitro, the final antioxidant activity of lipoic acid is determined by its concentration and by its antioxidant properties. Four antioxidant properties of lipoic acid have been studied: its metal chelating capacity, its ability to scavenge reactive oxygen species (ROS), its ability to regenerate endogenous antioxidants and its ability to repair oxidative damage. 4. Dihydrolipoic acid (DHLA), formed by reduction of lipoic acid, has more antioxidant properties than does lipoic acid. Both DHLA and lipoic acid have metal-chelating capacity and scavenge ROS, whereas only DHLA is able to regenerate endogenous antioxidants and to repair oxidative damage. 5. As a metal chelator, lipoic acid was shown to provide antioxidant activity by chelating Fe2+ and Cu2+; DHLA can do so by chelating Cd2+. 6. As scavengers of ROS, lipoic acid and DHLA display antioxidant activity in most experiments, whereas, in particular cases, pro-oxidant activity has been observed. However, lipoic acid can act as an antioxidant against the pro-oxidant activity produced by DHLA. 7. DHLA has the capacity to regenerate the endogenous antioxidants vitamin E, vitamin C and glutathione. 8. DHLA can provide peptide methionine sulfoxide reductase with reducing equivalents. This enhances the repair of oxidatively damaged proteins such as alpha-1 antiprotease. 9. Through the lipoamide dehydrogenase-dependent reduction of lipoic acid, the cell can draw on its NADH pool for antioxidant activity additionally to its NADPH pool, which is usually consumed during oxidative stress. 10. Within drug-related antioxidant pharmacology, lipoic acid is a model compound that enhances understanding of the mode of action of antioxidants in drug therapy.     

Rape flashbacks: constructing a new narrative

1. Radiotherapy has attracted increasing interest in recent years. It is known that ionizing radiation induces oxygen radical injury, whereas oxidative stress by the radiation can cause cellular responses to defense cellular injury. In this study, the metabolism of antioxidants in response to ionizing radiation to the brain was studied in the brain using experimental rabbits. 2. Ionizing radiation to the hemicerebrum caused an increase in the levels of glutathione (GSH) and the activity of a GSH synthesizing enzyme, gamma-glutamylcysteine synthetase (gamma-GCS), and Cu,Zn-superoxide dismutase (Cu,Zn-SOD). Ionizing radiation also induced DNA-damage estimated by the formation of 8-hydroxydeoxyguanosine. These changes were dependent on the radiation dose. 3. Previous intrathecal-administration of buthionine sulfoximine (100 microM), a specific inhibitor of gamma-GCS, increased DNA damage by radiation in the radiated hemicerebrum. That of S-methyl GSH, on the other hand, resulted in a significant reduction of DNA damage by radiation. 4. These results suggest that synthesis of GSH and Cu,Zn-SOD is responsive to ionizing radiation and this induction of antioxidants may play a role in reducing tissue damage in radiotherapy.     

An assessment of oxidative damage to proteins, lipids, and DNA in brain from patients with Alzheimer's disease

Oxidative stress may contribute to neuronal loss in Alzheimer's disease (AD). The present study compares the levels of oxidative damage to proteins, lipids, and DNA bases from seven different brain areas of AD and matched control tissues by using a range of techniques. No differences in levels of lipid peroxidation were found in any of the brain regions by using two different assay systems. Overall, there was a trend for protein carbonyl levels to be increased in AD in frontal, occipital, parietal, and temporal lobe, middle temporal gyrus, and hippocampus, but a significant difference was found only in the parietal lobe. Gas chromatography-mass spectrometry was used to measure products of damage to all four DNA bases. Increased levels of some (8-hydroxyadenine, 8-hydroxyguanine, thymine glycol, Fapy-guanine, 5-hydroxyuracil, and Fapy-adenine), but not all, oxidized DNA bases were observed in parietal, temporal, occipital, and frontal lobe, superior temporal gyrus, and hippocampus. The baseline level of oxidative DNA damage in the temporal lobe was higher than in other brain regions in both control and AD brain. The finding of increased oxidative damage to protein and DNA strengthens the possibility that oxidative damage may play a role in the pathogenesis of AD in at least some key brain regions.     

Increased nuclear DNA oxidation in the brain in Alzheimer's disease

Multiple lines of evidence indicate that oxidative stress is a contributor to neuronal death in Alzheimer's disease (AD). The oxidative damage that occurs to DNA may play a role in both normal aging and neurodegenerative diseases, including AD. This is a study of the oxidative damage that occurs in nuclear DNA in the brains of AD patients and cognitively intact, prospectively evaluated, age-matched control subjects. Nuclear DNA from frontal, temporal, and parietal lobes and cerebellum was isolated from 11 control subjects and 9 AD subjects, and oxidized purine and pyrimidine bases were quantitated using gas chromatography/mass spectrometry. Stable isotope-labeled oxidized base analogues were used as internal standards to measure 5-hydroxyuracil, 5-hydroxycytosine, 8-hydroxyadenine, 4,6-diamino-5-formamidopyrimidine (Fapy-adenine), 8-hydroxyguanine, and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy-guanine). Statistically significant elevations of 5-hydroxycytosine, 5-hydroxyuracil, 8-hydroxyadenine, and 8-hydroxyguanine were found in AD brain compared with control subjects (p < 0.05). There was an increased trend in the levels of Fapy-adenine in the AD brain, and Fapy-guanine showed a trend toward higher levels in control brains compared with AD. A generally higher level of oxidative DNA damage was present in neocortical regions than cerebellum. No significant correlation was observed between the oxidized bases and neurofibrillary tangle and senile plaque counts. Our results demonstrate that nuclear DNA damage by oxygen-derived radicals is increased in AD and support the concept that the brain is under increased oxidative stress in AD.     

Thoracic tumors in children with neurofibromatosis-1

Lymphocytes were isolated from volunteers before and after receiving a single supplement of vitamin C, vitamin E or beta-carotene. The lymphocytes were treated with H2O2, and DNA strand breaks were measured by single cell gel electrophoresis (the comet assay). Significant protection against oxidative DNA damage was evident 2-4 h after vitamin C intake, and 18-24 h after consumption of the other antioxidants. Lymphocytes from smokers were more sensitive to DNA damage than those from non-smokers, and they showed at least as great a protective effect with antioxidants.     

Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa

Reactive oxygen metabolites are known to disrupt sperm-oocyte fusion, sperm movement, and DNA integrity; however, the relative sensitivities of these elements to oxidative stress are unknown. In this study these factors were assessed in human spermatozoa exposed to increasing levels of oxidative stress achieved through the stimulation of endogenous oxidant generation with NADPH or direct exposure to hydrogen peroxide. At low levels of oxidative stress, DNA fragmentation was significantly reduced while the rates of sperm-oocyte fusion were significantly enhanced. As the level of oxidative stress increased, the spermatozoa exhibited significantly elevated levels of DNA damage (p < 0.001) and yet continued to express an enhanced capacity for sperm-oocyte fusion. At the highest levels of oxidative stress, extremely high rates of DNA fragmentation were observed but the spermatozoa exhibited a parallel loss in their capacities for movement and oocyte fusion. These studies emphasize how redox mechanisms can either enhance or disrupt the functional and genomic integrity of human spermatozoa depending on the intensity of the oxidative stimulus. Because these qualities are affected at different rates, spermatozoa exhibiting significant DNA damage are still capable of fertilizing the oocyte. These results may have long-term implications for the safety of assisted conception procedures in cases associated with oxidative stress.     

The inflammatory response system and the availability of plasma tryptophan in patients with primary sleep disorders and major depression

Food restriction increases maximal life span in rodents. Male rats that exercise in voluntary running wheels do not have an increase in maximal longevity despite a relative caloric deficit. In contrast, sedentary rats that are food restricted so as to cause the same caloric deficit have an extension of maximal longevity. It seemed possible that exercise-induced oxidative stress might prevent a maximum life span-extending effect of a caloric deficit to manifest itself. This study was done to determine if antioxidants would allow a maximal longevity-extending effect of exercise to manifest itself in male rats. The antioxidant diet had no effect on longevity of the runners (Antiox., 951 +/- 158 days versus control 937 + 171 days), or of the sedentary controls (875 +/- 127 versus 858 +/- 152 days). As in previous studies, wheel running modestly increased average longevity (approximately 9%), but had no effect on maximal life span. The finding that antioxidants had no effect on longevity of the wheel runners supports the interpretation that the caloric deficit induced by exercise in male rats does not have a life-extending effect that is countered by oxidative tissue damage.     

Oxidative damage induced by the fullerene C60 on photosensitization in rat liver microsomes

We have examined the ability of a commonly used fullerene, C60, to induce oxidative damage on photosensitization using rat liver microsomes as model membranes. When C60 was incorporated into rat liver microsomes in the form of its cyclodextrin complex and exposed to UV or visible light, it induced significant oxidative damage in terms of (1) lipid peroxidation as assayed by thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides and conjugated dienes, and (2) damage to proteins as assessed by protein carbonyls and loss of the membrane-bound enzymes. The oxidative damage induced was both time- and concentration-dependent. C60 plus light-induced lipid peroxidation was significantly inhibited by the quenchers of singlet oxygen ((1)O2), beta-carotene and sodium azide, and deuteration of the buffer-enhanced peroxidation. These observations indicate that C60 is an efficient inducer of peroxidation and is predominantly due to (1)O2. Biological antioxidants such as glutathione, ascorbic acid and alpha-tocopherol significantly differ in their ability to inhibit peroxidation induced by C60. Our studies, hence, indicate that C60, on photosensitization, can induce significant lipid peroxidation and other forms of oxidative damage in biological membranes and that this phenomenon can be greatly modulated by endogenous antioxidants and scavengers of reactive oxygen species.     

Alcohol and free radicals: from basic research to clinical prospects

An oxidative stress occurs in the liver of rats following various conditions of ethanol administration. The ethanol-inducible cytochrome P450 2E1 plays a key role in its generation, favoured itself by an increase in the "redox-active" fraction of intracellular non-heme iron. Administration of ethanol elicits the generation of the 1-hydroxyethyl radical, which has been identified in vivo. Its reactivity contributes to alcohol-induced immunological disturbances. Liver inflammatory and fibrotic disorders can be reproduced in rats by long-term ethanol administration associated with a high fat diet. The severity of these disorders is correlated to the intensity of the oxidative stress. Some conditions of ethanol administration to rats also elicit an oxidative stress in the myocardium and central nervous system. Through its inhibitory effect on glutamine synthetase activity and resulting excitotoxicity it may contribute to neuronal death and possibly to dependence on alcohol. Disorders related to an oxidative stress were also reported in the serum and erythrocytes as well as in liver biopsies from alcoholic individuals. Their detection may be useful to follow the evolution of alcoholic liver diseases. Supplementation with antioxidants such as vitamin E may be considered in the prevention of severe cellular disorders in individuals consuming large amounts of alcoholic beverages. An increase in free radical production is likely playing a role in the induction of severe cellular damage linked to repeated withdrawals occurring as a result of heavy and sporadic ethanol intake.     

Oxidative stress induces apoptosis in embryonic cortical neurons

Glutamate-induced glutathione depletion in immature embryonic cortical neurons has been shown to lead to oxidative stress and cell death. We have used this in vitro model to investigate the mechanism(s) by which free radicals induce neuronal degeneration. We find that glutathione depletion leads to hyper-condensation and fragmentation of chromatin into spherical or irregular shapes, a morphologic signature of apoptosis. These morphologic changes are accompanied by laddering of DNA into multiple oligonucleosomal fragments and can be prevented by the antioxidants idebenone and butylated hydroxyanisole. Cell death induced by glutathione depletion can also be prevented by inhibitors of macromolecular synthesis. Taken together, these observations suggest that oxidative stress can induce apoptosis in neurons.     

NF-κB regulates prenatal stress-induced cognitive impairment in offspring rats.

The study was designed to investigate whether nuclear factor of kappa B (NF-κB) plays regulating role in prenatal stress-induced cognitive impairment and oxidative damage in offspring rats. The authors used a rat model to study plasma levels of corticosterone and oxidative DNA damage (8-OH-dG), protein expression of P65/p50 NF-κB, and cognitive function in female and male offspring rats in middle pregnant stage and later pregnant stage. Prenatal stress affected the capability of learning and memory in the offspring, especially in later stage stressed female offspring. The levels of corticosterone and 8-OH-dG were enhanced in response to stress. Both middle and later stage stresses induced a significant decrease in P65 expression and a significant increase in P50 expression in female offspring. In addition, later stage stress induced a significant decrease in P50 expression in male offspring. These results suggest that NF-κB complex may be acting in a positive regulatory fashion in prenatal stress-induced cognitive impairment and that oxidative DNA damage may exacerbate the activation of NF-κB. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Increased 8-hydroxyguanine levels in DNA and its repair activity in rat kidney after administration of a renal carcinogen, ferric nitrilotriacetate

The renal carcinogen, ferric nitrilotriacetate (Fe-NTA), is known to induce oxidative stress and the subsequent formation of a type of oxidative DNA damage, 8-hydroxyguanine (8-OH-Gua), in the rat kidney (Umemura et al., 1990). Using an improved DNA isolation method (Nakae et al., 1995), which reduces the background level of 8-OH-Gua, we found a five-fold increase in the 8-OH-Gua level in kidney DNA after a single i.p. injection of Fe-NTA. On the basis of the report that 8-OH-Gua repair activity is enhanced after cells are exposed to oxidative stress due to ionizing radiation (Bases et al., 1992), the measurement of 8-OH-Gua repair activity will also be useful to assess cellular oxidative stress. The 8-OH-Gua repair enzyme activity was determined with an endonuclease assay using a 22 mer DNA that contains 8-OH-Gua at a specific position. A five-fold increase in the 8-OH-Gua repair activity as compared with the control, was observed in the target organ, the rat kidney, 120 h after Fe-NTA administration. In the non-target organ, the liver, the increase was not as large (two-fold). This simple assay of oxidative DNA damage repair will be useful for evaluating the carcinogenicity of oxygen radical forming chemicals, in addition to chemical analyses of oxidative DNA damage.     

Indicators of oxidative stress in aging rat brain. The effect of nerve growth factor

INTRODUCTION AND OBJECTIVE: Increased oxidative stress during ageing and the neurodegenerative disorders associated with this has been described. The central nervous system is particularly vulnerable to oxidative damage because of its high energy requirements, high oxygen consumption, high tissue concentration of iron and relatively low levels of some antioxidant systems. Treatment with neurotrophic factors may reverse neurone deterioration and stimulate cholinergic activity in aged rats. It may have a similar neuroprotector effect against damage due to ischaemic reperfusion, hypoglycaemia, inflammation and other pathological conditions involving oxidative stress. In this study we determined some indicators of oxidative stress in rat brains during ageing and evaluated this in response to a plan of treatment with murine nerve growth factor (FCN) for 38 days. MATERIAL AND METHODS: Biochemical techniques were used for determination of oxidative stress indicators. RESULTS AND CONCLUSIONS: We found that with age there was a significant increase in phospholipase A2 and superoxide dysmutase activity and concentration of hipoperoxidases, whilst the concentration of reduced glutathion fell. Catalase activity increased in the hippocampal and striate regions and decreased in the cortex and septal area. There was less oxidative stress in rats treated with FCN. In view of our results, we conclude that the level of oxidative stress increases with ageing, with significant differences between areas of the brain. The region most vulnerable to damage from species reactive to oxygen was the hippocampus, and the protective effect of FCN may be related to potentiation of antioxidant defenses.     

Haloacetate-induced oxidative damage to DNA in the liver of male B6C3F1 mice

Brominated and chlorinated haloacetates (HAs) are by-products of drinking water disinfection. Dichloroacetate (DCA) and trichloroacetate (TCA) are hepatocarcinogenic in rodents, but the brominated analogs have received little study. Prior work has indicated that acute doses of the brominated derivatives are more potent inducers of oxidative stress and increase the 8-hydroxydeoxyguanosine (8-OH-dG) content of the nuclear DNA in the liver. Since, DCA and TCA are also known as weak peroxisome proliferators, the present study was intended to determine whether this activity might be exacerbated by peroxisomal proliferation. Classical responses to peroxisome proliferators, cyanide-insensitive acyl-CoA oxidase activity and increased 12-hydroxylation of lauric acid, were elevated in a dose-related manner in mice maintained on TCA and clofibric acid (positive control), but not with DCA, dibromoacetate (DBA) or bromochloroacetate (BCA). Administration of the HAs in drinking water to male B6C3F1 mice for periods from 3 to 10 weeks resulted in dose-related increases in 8-OH-dG in nuclear DNA of the liver with DBA and BCA, but not with TCA or DCA. These findings indicate that oxidative damage induced by the haloacetates is, at least in part, independent of peroxisome proliferation. In addition, these data suggest that oxidative damage to DNA may play a more important role in the chronic toxicology of brominated compared to the chlorinated haloacetates.