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Oxidative damage and anti-oxidant capacity in two migratory bird species at a stop-over site.

We quantified in the garden warbler (Sylvia borin) and the barn swallow (Hirundo rustica), two long-distance migratory songbirds, the early oxidative damage (ROMs) and plasma anti-oxidant capacity (OXY) variation of individuals caught at a stop-over site after a sustained flight across the sea, during spring migration. Our main goal was to quantify the oxidative damage and anti-oxidant capacity variation in these two migratory species in relation to fat and muscle stores. The birds were sampled in Ponza, a small island along the migratory route of these species. The levels of ROMs and OXY did not show any differences between the two species and in general were higher in individuals with higher fat and protein stores. Nevertheless, the balance between ROMs and OXY was better in individuals in good condition. These patterns were similar in both species. No sex differences emerged for both ROMs and OXY in the barn swallow, the only species that could be sexed. Both markers of oxidative stress did not show any significant variation across a 30-min restrained experiment. These data are the first of this kind in wild birds in a migratory context and suggest that individuals in better condition are exposed to lower oxidative stress, providing an indirect evidence of the oxidative cost caused by prolonged flights.

Adipose Tissue↗

Oxidative damage of sulfur dioxide on various organs of mice: sulfur dioxide is a systemic oxidative damage agent.

Cu,Zn-superoxide dismutase (SOD), Se-dependent glutathione peroxidase (GSH-Px), catalase (CAT), and glutathione (GSH) play an important role in attenuating free radical-induced oxidative damage. The purpose of this research was to determine (1) whether sulfur dioxide (SO(2)) increases levels of lipid peroxidation and alters intracellular redox status in multiple organs of mice, and (2) whether SO(2) is a systemic toxic agent. The effect of SO(2) on levels of thiobarbituric acid-reactive substances (TBARS) and GSH and activities of SOD, GSH-Px, and CAT were investigated in nine organs (brain, lung, heart, liver, stomach, intestine, spleen, kidney, and testis) of Kunming albino mice of both sexes. SO(2) at 20 ppm (56 mg/m(3)) was administrated to the animals of SO(2) groups in an exposure chamber for 6 h/day for 7 days while control groups were exposed to filtered air in the same condition. Results show that SO(2) inhalation decreased significantly activities of SOD and GSH-Px in all organs tested in all SO(2) groups, with respect to their corresponding control groups; CAT activities in all organs tested of both sexual mice were significantly unaltered, except CAT activities in livers were significantly lowered by SO(2); SO(2) exposure decreased significantly GSH contents and significantly increased TBARS levels of all organs tested, in comparison with their respective control groups. These results lead to two conclusions: (1) SO(2) is a systemic oxidative damage agent. It results in a significant increase in the lipid peroxidation process in all organs tested of mice of both sexes, which is accompanied by changes of antioxidant status in these organs. (2) SO(2) may cause toxicological damage to multiple organs of animals, and it is suggested that the oxidative damage produced by SO(2) inhalation may influence or promote the progression or occurrence of some disease states of various organs, not only to respiratory system. Further work is required to understand the toxicological role of SO(2) on multiple or even all organs in mammals.

Air Pollutants↗

Oxidative damage in the senescence-accelerated mouse.

The senescence-accelerated mouse (SAM) exhibited a shortened life span (about 18 months) and early manifestation of various signs of senescence, including changes in physical activity, skin, and spinal curvature. The mechanism of senescence acceleration in SAM is thought to be related to free radical damage. Oxidative phosphorylation was estimated in liver mitochondria from SAMPS and the senescence-resistant subtrain, SAMR1. The respiratory control ratio decreased during aging, and the ATP/O, an index of ATP synthesis, was depressed at 18 months of age in SAMPS. DNP-dependent uncoupled respiration in liver mitochondria was markedly decreased, and active uptake of calcium was markedly dysfunctional with aging. These findings suggest that the functional disorders in mitochondria may be closely related to the shorter life span of SAMPS. White-footed (WF) mice can live at least to 5.5 years, when some animals are still capable of reproducing and their external body condition remains healthy. The mitochondrial functions were examined in the same way as in the SAM experiments. However, no particular finding responsible for their longevity was observed in WF mice at 3 and 12 months old. More comprehensive examinations on more aged WF mice are needed for explanation of their greater longevity.

Aging↗

DNA damage and susceptibility to oxidative damage in lymphocytes: effects of carotenoids in vitro and in vivo.

Reports on the effects of carotenoids are conflicting. The present paper examines similarities and differences from contiguous studies in vitro and in vivo. Single-cell gel electrophoresis was used to measure the frequency of single-strand breaks (SSB) in the cell line MOLT-17 (as a model system) and human peripheral blood lymphocytes (PBL). MOLT-17 cells were supplemented with beta-carotene, lutein or lycopene at a range of concentrations (0.00-8.00 micromol/l) using a liposome delivery method. Uptake was dose-dependent. beta-Carotene concentration in the media had no effect on SSB in control cells, but incubation with lycopene or lutein (>2.00 micromol/l) increased the numbers of SSB in control cells. MOLT-17 DNA was less susceptible to oxidative damage (100 micromol H2O2/l, 5 min, 4 degrees C) following incubation with carotenoids between 0.50 and 1.00 micromol/l; at >1.00 micromol/l the effects were ambiguous. Apparently healthy male volunteers supplemented their habitual diets with lutein, beta-carotene or lycopene (natural isolate capsules, 15 mg/d, 4 weeks) in three independent studies, raising plasma concentrations to different extents. Lycopene and lutein had no effect on SSB in control PBL or following oxidative challenge. However, increased plasma beta-carotene was associated with more SSB in control cells whilst PBL DNA resistance to oxidative damage ex vivo was unaffected. These results suggest that the carotenoids are capable of exerting two overlapping but distinct effects: antioxidant protection by scavenging DNA-damaging free radicals and modulation of DNA repair mechanisms.

Adolescent↗

The oxidative damage of plasmid DNA by ascorbic acid derivatives in vitro: the first research on the relationship between the structure of ascorbic acid and the oxidative damage of plasmid DNA.

To study the structure-function relationship of the oxidative-damage effect of ascorbic acid, we have focused on the interaction between plasmid DNA pUC19 and a series of ascorbic acid derivatives modified on different OH groups in the presence of transition metal ions. Some ascorbic acid derivatives can selectively cleave plasmid DNA from Form I to Form II in the presence of low concentration of Cu2+ just like ascorbic acid itself, while other derivatives oxidatively damage plasmid DNA slightly. We found that those derivatives with unattached 2-OH and 3-OH groups retain the ability to cleave the plasmid DNA. The derivatives that have been methylated on 2-OH or 3-OH can only cleave plasmid DNA softly, and those derivatives that have been protected on both 2-OH and 3-OH can hardly exert an oxidative damage on plasmid DNA under the same condition. Form these results, we can draw the conclusion that 2-OH and 3-OH groups of the ascorbic acid molecule contribute most to this biological activity.

Ascorbic Acid↗

Tributyltin induces DNA damage as well as oxidative damage in rats.

In this study, oxidative and DNA damage were measured synchronously after Sprague-Dawley rats were exposed to different dosages of tributyltin (TBT) for 3 and 7 consecutive days. Oxidative damage was measured by analyzing the production of hepatic reactive oxygen species (ROS), the activity of superoxide dismutase (SOD), and the content of malondialdehyde (MDA). DNA damage was measured by single-cell gel electrophoresis (comet assay). After 3 days of exposure, significant differences in ROS production could only be seen between the control and the highest dosage group (10 mg/kg BW d), although after 7 days of treatment, ROS production increased in a dose-dependent manner. SOD activity increased with dosage after 3 days of exposure and decreased with dosage after 7 days of exposure. TBT also induced significant production of MDA after 7 days of exposure. The changes in ROS, SOD, and MDA found in this study suggest that the antioxidative systems of rats were activated by TBT in the first 3 days of exposure but had become exhausted by 7 days of exposure. In the comet assay, the number of cells with damaged DNA in rats treated with TBT increased with dosage of TBT. The most likely mechanism of the DNA breakage induced by TBT is oxidative damage. It can be concluded that exposure of TBT can promote both oxidative and DNA damage in mammals in vivo.

Animals↗

Extracts of Ginkgo biloba and Panax ginseng protect brain proteins from free radical induced oxidative damage in vitro.

Oxidative damage to normal human brain tissue was induced following exposure to hydroxyl (OH.) or superoxide (O2-.) free radical species generated by CO60 irradiation in vitro. Both enzymic and cytoskeletal proteins showed substantial (dose dependent) oxidative damage following exposure to OH. or O2-., as quantified by SDS-polyacrylamide gel electrophoretic analysis. Extracts of Ginkgo biloba or Panax ginseng showed a remarkable capacity to protect brain tissue proteins from oxidative damage in vitro, even at extreme (2000 kRads) dosage levels of OH. or O2-.. We suggest, therefore, that the beneficial effect of these plant extracts in preventing brain tissue damage in vivo (e.g. following ischemia-reperfusion) may result from their action in protecting brain proteins from oxidative damage, in addition to their previously reported capacity to reduce free radical induced lipid peroxidation.

Brain Damage, Chronic↗

Effect of proteasome inhibition on cellular oxidative damage, antioxidant defences and nitric oxide production.

The ubiquitin/proteasome pathway plays an essential role in protein turnover in vivo, and contributes to removal of oxidatively damaged proteins. We examined the effects of proteasome inhibition on viability, oxidative damage and antioxidant defences in NT-2 and SK-N-MC cell lines. The selective proteasome inhibitor, lactacystin (1 microM) caused little loss of viability, but led to significant increases in levels of oxidative protein damage (measured as protein carbonyls), ubiquitinated proteins, lipid peroxidation and 3-nitrotyrosine, a biomarker of the attack of reactive nitrogen species (such as peroxynitrite, ONOO(-)) upon proteins. Higher levels (25 microM) of lactacystin did not further increase the levels of carbonyls, lipid peroxidation, 3-nitrotyrosine, or ubiquitinated proteins, but produced increases in the levels of 8-hydroxyguanine (a biomarker of oxidative DNA damage) and falls in levels of GSH. Lactacystin (25 microM) caused loss of viability, apparently by apoptosis, and also increased production of nitric oxide (NO.) (measured as levels of NO2- plus NO3-) by the cells; this was inhibited by N-nitro-L-arginine methyl ester (L-NAME), which also decreased cell death induced by 25 microM lactacystin and decreased levels of 3-nitrotyrosine. The NO. production appeared to involve nNOS; iNOS or eNOS were not detectable in either cell type. Another proteasome inhibitor, epoxomicin, had similar effects.

Acetylcysteine↗

Resonance Raman and electron paramagnetic resonance studies on oxidized and ferricyanide-treated Clostridium pasteurianum ferredoxin. Vibrational assignments from 34S shifts and evidence for conversion of 4 to 3 iron-sulfur clusters via oxidative damage. Vibrational assignments from 34S shifts and evidence for conversion of 4 to 3 iron-sulfur clusters via oxidative damage.

Resonance Raman spectra are reported for oxidized ferredoxin from Clostridium pasteurianum and for protein reconstituted with 34S2-, using 4579 A laser excitation. The spectra are of much higher quality than that previously reported, and the 34S shifts provide assignments of the Fe-S modes. After treatment with ferricyanide, the resonance Raman spectrum closely resembles that of the [3Fe-3S] protein, ferredoxin II from D. gigas; the 34S shifts aid in assignments of the [3Fe-3S] modes. The epr signal associated with the [3Fe-3S] cluster (g = 2.01) corresponds maximally to 0.80 spin/molecule. Anaerobic addition of excess sulfide to the reduced, ferricyanide-treated protein regenerates a [4Fe-4S]1+ epr spectrum, equivalent in intensity to the [3Fe-3S] signal. The ubiquitous occurrence of a g = 2.01 signal in preparations of [4Fe-4S] proteins can be attributed to variable amounts of [3Fe-3S] cluster generated by adventitious oxidation. The ready conversion of [4Fe-4S] to [3Fe-3S] clusters in C. pasterianum raises the possibility that some [3Fe-3S] proteins may actually arise by oxidative damage of [4Fe-4S] proteins during aerobic isolation.

Clostridium↗

Oxidative damage by ozone and nitrogen dioxide: synergistic toxicity in vivo but no evidence of synergistic oxidative damage in an extracellular fluid.

Inhalation of ozone (O3) and/or nitrogen dioxide (.NO2) is associated with the development of inflammation in the respiratory tract and various alterations in pulmonary functions. Respiratory tract lining fluids (RTLFs) represent the first biological fluids coming into contact with these inhaled toxicants. Using plasma as a surrogate for RTLFs, we have previously shown that O3 [Cross, Motchnik, Bruener, Jones, Kaur, Ames and Halliwell (1992) FEBS Lett. 298, 269-272] and .NO2 [Halliwell, Hu, Louie, Duvall, Tarkington, Motchnik and Cross (1992) FEBS Lett. 313, 62-66] are both capable of depleting antioxidants and damaging proteins and lipids. O3 particularly damages proteins, whereas .NO2 induces the peroxidation of lipids and nitrates aromatic amino acids. It has been reported that O3 and .NO2 cause synergistic toxicity in rodents [Gielzleichter, Witschi and Last (1992) Tox. Appl. Pharmacol. 116, 1-9]. In the present chapter, we review evidence showing that combined exposure of these two oxidant gases to human plasma fails to exert synergistic oxidative damage to plasma constituents, and in fact, O3 and .NO2 antagonize each other's actions. We conclude that the potentiating effect of these two gases on morbidity and mortality in rodents represents a complex interactive biological effect rather than a simple synergistic oxidative effect in extracellular fluids.

Animals↗

Can antioxidant vitamins materially reduce oxidative damage in humans?

Endogenous oxidative damage to proteins, lipids, and DNA is thought to be an important etiologic factor in aging and the development of chronic diseases such as cancer, atherosclerosis, and cataract formation. The pathology associated with these diseases is likely to occur only after the production of reactive oxygen species has exceeded the body's or cell's capacity to protect itself and effectively repair oxidative damage. Vitamin C, vitamin E, and beta-carotene, often referred to as "antioxidant vitamins," have been suggested to limit oxidative damage in humans, thereby lowering the risk of certain chronic diseases. However, epidemiological studies and clinical trials examining the efficacy of antioxidant vitamins, either individually or in combination, to affect disease outcome rarely address possible underlying mechanisms. Thus, in these studies it is often assumed that antioxidant vitamins act by lowering oxidative damage, but evidence in support of this contention is not provided. Therefore, in this review, we examine the scientific evidence that supplementation of humans with vitamin C, vitamin E, or beta-carotene lowers in vivo oxidative damage to lipids, proteins, or DNA based on the measurement of oxidative biomarkers, not disease outcome. With the only exception of supplemental vitamin E, and possibly vitamin C, being able to significantly lower lipid oxidative damage in both smokers and nonsmokers, the current evidence is insufficient to conclude that antioxidant vitamin supplementation materially reduces oxidative damage in humans.

Animals↗

Red blood cells contain a pathway for the degradation of oxidant-damaged hemoglobin that does not require ATP or ubiquitin.

It is generally accepted that ATP is required for intracellular protein breakdown. Reticulocytes contain a soluble ATP-dependent pathway for the degradation of highly abnormal proteins and for the elimination of certain proteins during cell maturation. Reticulocytes and erythrocytes also selectively degrade proteins damaged by oxidation. When these cells were exposed to oxidants, such as phenylhydrazine or nitrite, they showed a large increase in protein breakdown. This oxidant-induced proteolysis was not inhibited in cells depleted of ATP. However, ATP depletion did prevent the degradation of pre-existent cell proteins. In reticulocyte extracts, phenylhydrazine-treated hemoglobin is also degraded rapidly by an ATP-independent process, unlike endogenous proteins and many exogenous polypeptides. This lack of an ATP requirement means that the degradation of oxidant-damaged proteins does not require ligation to ubiquitin (even though phenylhydrazine treatment does make hemoglobin a very good substrate for ubiquitin conjugation). In many respects, the pathway for breakdown of oxidant-treated hemoglobin differs from the ATP-dependent process. The latter has a much higher activation energy than the degradation of oxidized proteins. The ATP-dependent process is inhibited by hemin, 3,4-dichloroisocoumarin, diisopropylfluorophosphate and N-ethylmaleimide. The ATP-independent pathway is less sensitive to N-ethylmaleimide, hemin, and 3,4-dichloroisocoumarin and is not affected by diisopropylfluorophosphate. In addition, only the ATP-dependent proteolytic process is inactivated by dilution or incubation at 37 degrees C in the absence of nucleotides. Reticulocytes thus contain multiple soluble systems for degrading proteins and can rapidly hydrolyze certain types of abnormal proteins by either an ATP-independent or ATP-dependent process. Erythrocytes lack the ATP-dependent process present in reticulocytes; however, erythrocytes retain the capacity to degrade oxidant-damaged hemoglobin. These two processes probably are active in the elimination of different types of abnormal proteins.

2,4-Dinitrophenol↗

Properties and biological functions of the NTH and FPG proteins of Escherichia coli: two DNA glycosylases that repair oxidative damage in DNA.

Oxidative damage to DNA is one of the most important causes of spontaneous mutations and may play a role in aging and related diseases, such as cancer, in humans. Oxidative damage results from the attack of biomolecules by free radicals and reactive oxygen species formed as byproducts of normal cell metabolism or during oxidative stress. To counteract the lethal and mutagenic effects of oxidative lesions in DNA, cells have developed defence strategies including DNA repair systems. In Escherichia coli, the repair of oxidized bases in DNA is mostly mediated by the base excision repair pathway. The first step in this DNA repair pathway is catalysed either by the NTH protein which excises oxidized pyrimidines or by the FPG protein which excises oxidized purines. The nucleotide excision repair pathway mediated by the UvrABC complex may also play a role when the DNA glycosylases are inactive or saturated. This review summarizes the structural and catalytic properties of the NTH and FPG proteins of Escherichia coli and presents evidence to indicate that these two enzymes constitute an important component of the cellular defence against oxidative stress in prokaryotes and eukaryotes.

Amino Acid Sequence↗

Superoxide dismutase 1 protects retinal cells from oxidative damage.

Bolstering the endogenous oxidative damage defense system is a good strategy for development of treatments to combat neurodegenerative diseases in which oxidative damage plays a role. A first step in such treatment development is to determine the role of various components of the defense system in cells that degenerate. In this study, we sought to determine the role of superoxide dismutase 1 (SOD1) in two models of oxidative damage-induced retinal degeneration. In one model, paraquat is injected into the vitreous cavity and then enters retinal cells and generates reactive oxygen species (ROS) that cause progressive retinal damage. Assessment of retinal function with serial electroretinograms (ERGs) showed that sod1 -/- mice were much more sensitive than sod1 +/+ mice to the damaging effects of paraquat, while sod1 +/- mice showed intermediate sensitivity. Compared to sod1 +/+ mice, sod1 -/- mice showed greater paraquat-induced oxidative damage and apoptosis. In the second model, mice were exposed to hyperoxia for several weeks, and sod1 -/- mice showed significantly greater reductions in ERG amplitudes than sod1 +/+ mice. In both of these models, transgenic mice carrying a sod1 transgene driven by a beta-actin promoter showed less oxidative stress-induced reduction in ERG amplitudes. These data demonstrate that SOD1 protects retinal cells against paraquat- and hyperoxia-induced oxidative damage and suggest that overexpression of SOD1 should be considered as one component of ocular gene therapy to prevent oxidative damage-induced retinal degeneration.

Animals↗

Long-range oxidative damage in DNA/RNA duplexes.

Oxidative damage as a result of DNA-mediated long-range charge transport occurs readily and at high yield in duplex DNA, and it is of interest whether similar damage can occur in duplex oligonucleotides that include both ribo- and deoxyribonucleotides. Assemblies containing RNA and mixed RNA.DNA strands were constructed containing tethered ethidium as a photooxidant. In photooxidation experiments, long-range oxidative damage to the ribose-containing strand of the oligonucleotide duplexes was examined. Hole injection by photoexcited ethidium followed by radical migration to oxidatively susceptible guanines afforded significant damage on ribose-containing strands at long range ( approximately 35 A). This damage does not differ substantially in yield and location from that found in B-DNA duplexes. No oxidative damage was found upon photooxidation of DNA/RNA duplexes containing tethered metallointercalator, despite the ability of the rhodium complex to promote oxidative damage at a distance in DNA duplexes. This result is attributed to the poor coupling of the rhodium complex into the A-like RNA/DNA duplex. The ability for long-range charge transport to occur in double-stranded nucleic acids of different comformations is considered in light of modeling studies that show interstrand base-base overlap between the opposing, complementary strands that make up RNA/DNA hybrid duplexes. Thus, the possibility of long-range radical migration to effect oxidative damage or signaling may be considered also in the context of transcriptional events.

Base Composition↗

SVPD-post-labeling detection of oxidative damage negates the problem of adventitious oxidative effects during 32P-labeling.

The exploitation of oxidative DNA lesions as biomarkers of oxidative stress in vivo requires techniques that allow for the precise and valid measurement of oxidative damage to DNA. Previously, endogenous levels of the oxidative lesion 8-hydroxy-2'-deoxyguanosine (8-HO-dG) in rat tissues determined by a micrococcal nuclease/calf spleen phosphodiesterase-based 32P-post-labeling protocol were found to be at least 10-fold higher than those determined by HPLC with electrochemical detection. This was attributed to the adventitious oxidation of the normal nucleotides (dGp) occurring during the labeling stage of the postlabeling protocol, which could only be prevented by the introduction of additional chromatographic steps to remove the unmodified species prior to labeling. In the present study we report that an alternative snake venom phosphodiesterase-based 32P-post-labeling procedure (SVPD-postlabeling) negates the problem of adventitious oxidative damage during labeling by virtue of a unique digestion strategy. In SVPD-post-labeling, digestion yields certain lesions (thymine glycols, phosphoglycolates and abasic sites) as damage-containing dimer species which are ready substrates for labeling. In contrast, the undamaged DNA is recovered as mononucleoside species (dN) which are not substrates for labeling and so remain undetected. Furthermore, even if the mononucleosides are oxidized during labeling, they will not contribute to the level of damage detected. Indeed, we demonstrate that neither the external gamma-irradiation of the digested DNA samples nor increasing the incubation time of the labeling reaction alters the levels of damage detected by SVPD-post-labeling. The negation of adventitious oxidative effects during labeling deems that an optimized SVPD-post-labeling procedure should be well-suited for the biomonitoring of endogenous oxidative stress in vivo.

Animals↗