Ascorbate removes key precursors to oxidative damage by cell-free haemoglobin in vitro and in vivo

Haemoglobin initiates free radical chemistry. In particular, the interactions of peroxides with the ferric (met) species of haemoglobin generate two strong oxidants: ferryl iron and a protein-bound free radical. We have studied the endogenous defences to this reactive chemistry in a rabbit model following 20% exchange transfusion with cell-free haemoglobin stabilized in tetrameric form [via cross-linking with bis-(3,5-dibromosalicyl)fumarate]. The transfusate contained 95% oxyhaemoglobin, 5% methaemoglobin and 25 microM free iron. EPR spectroscopy revealed that the free iron in the transfusate was rendered redox inactive by rapid binding to transferrin. Methaemoglobin was reduced to oxyhaemoglobin by a slower process (t(1/2) = 1 h). No globin-bound free radicals were detected in the plasma. These redox defences could be fully attributed to a novel multifunctional role of plasma ascorbate in removing key precursors of oxidative damage. Ascorbate is able to effectively reduce plasma methaemoglobin, ferryl haemoglobin and globin radicals. The ascorbyl free radicals formed are efficiently re-reduced by the erythrocyte membrane-bound reductase (which itself uses intra-erythrocyte ascorbate as an electron donor). As well as relating to the toxicity of haemoglobin-based oxygen carriers, these findings have implications for situations where haem proteins exist outside the protective cell environment, e.g. haemolytic anaemias, subarachnoid haemorrhage, rhabdomyolysis.     

Effect of thyroid state on susceptibility to oxidants and swelling of mitochondria from rat tissues

The effects of the thyroid state on oxidative damage, antioxidant capacity, susceptibility to in vitro oxidative stress and Ca(2+)-induced permeabilization of mitochondria from rat tissues (liver, heart, and gastrocnemious muscle) were examined. Hypothyroidism was induced by administering methimazole in drinking water for 15 d. Hyperthyroidism was elicited by a 10 d treatment of hypothyroid rats with triiodothyronine (10 micro g/100 g body weight). Mitochondrial levels of hydroperoxides and protein-bound carbonyls significantly decreased in hypothyroid tissues and were reported above euthroid values in hypothyroid rats after T(3) treatment. Mitochondrial vitamin E levels were not affected by changes of animal thyroid state. Mitochondrial Coenzyme Q9 levels decreased in liver and heart from hypothyroid rats and increased in all hyperthyroid tissues, while Coenzyme Q10 levels decreased in hypothyroid liver and increased in all hyperthyroid tissues. The antioxidant capacity of mitochondria was not significantly different in hypothyroid and euthyroid tissues, whereas it decreased in the hyperthyroid ones. Susceptibility to in vitro oxidative challenge decreased in mitochondria from hypothyroid tissues and increased in mitochondria from hyperthyroid tissues, while susceptibility to Ca(2+)-induced swelling decreased only in hypothyroid liver mitochondria and increased in mitochondria from all hyperthyroid tissues. The tissue-dependence of the mitochondrial susceptibility to stressful conditions in altered thyroid states can be explained by different thyroid hormone-induced changes in mitochondrial ROS production and relative amounts of mitochondrial hemoproteins and antioxidants. We suggest that susceptibilities to oxidants and Ca(2+)-induced swelling may have important implications for the thyroid hormone regulation of the turnover of proteins and whole mitochondria, respectively.     

Effect of cold-induced hyperthyroidism on H2O2 production and susceptibility to stress conditions of rat liver mitochondria

Previous studies have shown that T3 treatment and cold exposure induce similar biochemical changes predisposing rat liver to oxidative stress. This suggests that the liver oxidative damage observed in experimental and functional hyperthyroidism is mediated by thyroid hormone. To support this hypothesis we investigated whether middle-term cold exposure (2 and 10 days), like T3 treatment, also increases H2O2 release by liver mitochondria. We found that the rate of H2O2 release increased only during State 4 respiration, but faster flow of reactive oxygen species (ROS) from mitochondria to the cytosolic compartment was ensured by the concomitant increase in tissue mitochondrial proteins. Cold exposure also increased the capacity of mitochondria to remove H2O2. This indicates that cold causes accelerated H2O2 production, which might depend on enhanced autoxidizable carrier content and should lead to increased mitochondrial damage. Accordingly, mitochondrial levels of hydroperoxides and protein-bound carbonyls were higher after cold exposure. Levels of low-molecular weight antioxidants were not related to the extent of oxidative damage, but susceptibility to both in vitro oxidative challenge and Ca2+-induced swelling increased in mitochondria from cold exposed rats. The cold-induced changes in several parameters, including susceptibility to swelling, were time dependent, because they were apparent or greater after 10 days cold exposure. The cold-induced increase in swelling may be a feedback mechanism to limit tissue oxidative stress, purifying the mitochondrial population from ROS-overproducing mitochondria, and the time course for such change is consistent with the gradual development of cold adaptation.     

Effect of vitamin C on lipid hydroperoxides and carbonyl groups content of rat plasma depending on age and acute heat exposure

Free radicals produced during hyperthermic stress and aging are thought to play an important role in the degenerative process. To investigate the correlation between oxidative damages caused by acute heat exposure or aging, and the protective effect of vitamin C in vivo, we determined the levels of oxidative protein damage, lipid peroxidation, content of endogenous ascorbic acid, and glutathione in the plasma of young and old Wistar rats, subjected or not-subjected to acute heat stress. The results showed that the level of oxidative protein damage (measured as carbonyl content) in plasma was significantly higher in elderly and in heat-exposed animals. Vitamin C treatment led to inhibition on carbonyl production much more pronounced in young heat-exposed than in aged heat-exposed rats. Aging and acute heat exposure correlated positively with increased production of lipid hydroperoxides in rats plasma, but there were no significant differences in lipid hydroperoxides levels between young and old heat-exposed rats, depending on the treatment with vitamin C. Multiple backward regression analysis showed ascorbic acid to be the only determining variable of lipid hydroperoxides levels in unexposed rats. It was concluded that aging and heat exposure instigate an increase of lipid peroxidation and protein oxidation in rat plasma, while vitamin C supplementation significantly counteracts these changes.     

Honest sexual signalling mediated by parasite and testosterone effects on oxidative balance

Extravagant ornaments evolved to advertise their bearers'' quality, the honesty of the signal being ensured by the cost paid to produce or maintain it. The oxidation handicap hypothesis (OHH) proposes that a main cost of testosterone-dependent ornamentation is oxidative stress, a condition whereby the production of reactive oxygen and nitrogen species (ROS/RNS) overwhelms the capacity of antioxidant defences. ROS/RNS are unstable, very reactive by-products of normal metabolic processes that can cause extensive damage to key biomolecules (cellular proteins, lipids and DNA). Oxidative stress has been implicated in the aetiology of many diseases and could link ornamentation and genetic variation in fitness-related traits. We tested the OHH in a free-living bird, the red grouse. We show that elevated testosterone enhanced ornamentation and increased circulating antioxidant levels, but caused oxidative damage. Males with smaller ornaments suffered more oxidative damage than those with larger ornaments when forced to increase testosterone levels, consistent with a handicap mechanism. Parasites depleted antioxidant defences, caused oxidative damage and reduced ornament expression. Oxidative damage extent and the ability of males to increase antioxidant defences also explained the impacts of testosterone and parasites on ornamentation within treatment groups. Because oxidative stress is intimately linked to immune function, parasite resistance and fitness, it provides a reliable currency in the trade-off between individual health and ornamentation. The costs induced by oxidative stress can apply to a wide range of signals, which are testosterone-dependent or coloured by pigments with antioxidant properties.     

Role of thioredoxins in the response of Saccharomyces cerevisiae to oxidative stress induced by hydroperoxides

Glutaredoxins and thioredoxins are highly conserved, small, heat-stable oxidoreductases. The yeast Saccharomyces cerevisiae contains two gene pairs encoding cytoplasmic glutaredoxins (GRX1, GRX2) and thioredoxins (TRX1, TRX2), and we have used multiple mutants to determine their roles in mediating resistance to oxidative stress caused by hydroperoxides. Our data indicate that TRX2 plays the predominant role, as mutants lacking TRX2 are hypersensitive, and mutants containing TRX2 are resistant to these oxidants. However, the requirement for TRX2 is only apparent during stationary phase growth, and we present three lines of evidence that the thioredoxin isoenzymes actually have redundant activities as antioxidants. First, the trx1 and trx2 mutants show wild-type resistance to hydroperoxide during exponential phase growth; secondly, overexpression of either TRX1 or TRX2 leads to increased resistance to hydroperoxides; and, thirdly, both Trx1 and Trx2 are equally able to act as cofactors for the thioredoxin peroxidase, Tsa1. The antioxidant activity of thioredoxins is required for both the survival of yeast cells as well as protection against oxidative stress during stationary phase growth, and correlates with an increase in the expression of both TRX1 and TRX2. We show that the requirement for thioredoxins during this growth phase is dependent on their activity as cofactors for the antioxidant enzyme Tsa1, and for regulation of the redox state and protein-bound levels of the low-molecular-weight antioxidant glutathione.     

Mitochondrial oxidative stress plays a key role in aging and apoptosis

Harman first suggested in 1972 that mitochondria might be the biological clock in aging, noting that the rate of oxygen consumption should determine the rate of accumulation of mitochondrial damage produced by free radical reactions. Later in 1980 Miquel and coworkers proposed the mitochondrial theory of cell aging. Mitochondria from postmitotic cells use O2 at a high rate, hence releasing oxygen radicals that exceed the cellular antioxidant defences. The key role of mitochondria in cell aging has been outlined by the degeneration induced in cells microinjected with mitochondria isolated from fibroblasts of old rats, especially by the inverse relationship reported between the rate of mitochondrial production of hydroperoxide and the maximum life span of species. An important change in mitochondrial lipid composition is the age-related decrease found in cardiolipin content. The concurrent enhancement of lipid peroxidation and oxidative modification of proteins in mitochondria further increases mutations and oxidative damage to mitochondrial DNA (mtDNA) in the aging process. The respiratory enzymes containing the defective mtDNA-encoded protein subunits may increase the production of reactive oxygen species, which in turn would aggravate the oxidative damage to mitochondria. Moreover, superoxide radicals produced during mitochondrial respiration react with nitric oxide inside mitochondria to yield damaging peroxynitrite. Treatment with certain antioxidants, such as sulphur-containing antioxidants, vitamins C and E, or the Ginkgo biloba extract EGb 761, protects against the age-associated oxidative damage to mtDNA and the oxidation of mitochondrial glutathione. Moreover, the EGb 761 extract also prevents changes in mitochondrial morphology and function associated with aging of the brain and liver.     

The free-radical damage theory: Accumulating evidence against a simple link of oxidative stress to ageing and lifespan

Recent work on a small European cave salamander (Proteus anguinus) has revealed that it has exceptional longevity, yet it appears to have unexceptional defences against oxidative damage. This paper comes at the end of a string of other studies that are calling into question the free-radical damage theory of ageing. This theory rose to prominence in the 1990s as the dominant theory for why we age and die. Despite substantial correlative evidence to support it, studies in the last five years have raised doubts over its importance. In particular, these include studies of mice with the major antioxidant genes knocked out (both singly and in combination), which show the expected elevation in oxidative damage but no impact on lifespan. Combined, these findings raise fundamental questions over whether the free-radical damage theory remains useful for understanding the ageing process, and variation in lifespan and life histories.     

Taurine supplementation decreases oxidative stress in skeletal muscle after eccentric exercise

Infrequent exercise, typically involving eccentric actions, has been shown to cause oxidative stress and to damage muscle tissue. High taurine levels are present in skeletal muscle and may play a role in cellular defences against free radical‐mediated damage. This study investigates the effects of taurine supplementation on oxidative stress biomarkers after eccentric exercise (EE). Twenty‐four male rats were divided into the following groups (n = 6): control; EE; EE plus taurine (EE + Taurine); EE plus saline (EE + Saline). Taurine was administered as a 1‐ml 300 mg kg^?1 per body weight (BW) day^?1 solution in water by gavage, for 15 consecutive days. Starting on the 14th day of supplementation, the animals were submitted to one 90‐min downhill run session and constant velocity of 1·0 km h^?1. Forty‐eight hours after the exercise session, the animals were killed and the quadriceps muscles were surgically removed. Production of superoxide anion, creatine kinase (CK) levels, lipoperoxidation, carbonylation, total thiol content and antioxidant enzyme were analysed. Taurine supplementation was found to decrease superoxide radical production, CK, lipoperoxidation and carbonylation levels and increased total thiol content in skeletal muscle, but it did not affect antioxidant enzyme activity after EE. The present study suggests that taurine affects skeletal muscle contraction by decreasing oxidative stress, in association with decreased superoxide radical production. Copyright © 2010 John Wiley & Sons, Ltd.     

Epr Studies on the Effects of Complexation of Heme by Hemopexin upon its Reactions with Organic Peroxides

Hemopexin, a heme-binding serum glycoprotein, is thought to play an important role in the prevention of oxidative damage that may be catalysed by free heme. Through the use of EPR techniques, the generation of free radicals from organic hydroperoxides by heme and heme-hemopexin complexes, and the concomitant formation of high oxidation-state iron species has been studied; these species are implicated as causative agents in processes such as cardiovascular disease and carcinogenesis. From the rates of production of these species from both n-alkyl and branched hydroperoxides, it has been inferred that the dramatic reduction in the yield of oxidising species generated by heme upon its complexation with hemopexin arises from steric hindrance of the access of hydroperoxide to the bound heme.     

Induced defences in marine and freshwater phytoplankton: a review

Many organisms have developed defences to avoid predation by species at higher trophic levels. The capability of primary producers to defend themselves against herbivores affects their own survival, can modulate the strength of trophic cascades and changes rates of competitive exclusion in aquatic communities. Algal species are highly flexible in their morphology, growth form, biochemical composition and production of toxic and deterrent compounds. Several of these variable traits in phytoplankton have been interpreted as defence mechanisms against grazing. Zooplankton feed with differing success on various phytoplankton species, depending primarily on size, shape, cell wall structure and the production of toxins and deterrents. Chemical cues associated with (i) mechanical damage, (ii) herbivore presence and (iii) grazing are the main factors triggering induced defences in both marine and freshwater phytoplankton, but most studies have failed to disentangle the exact mechanism(s) governing defence induction in any particular species. Induced defences in phytoplankton include changes in morphology (e.g. the formation of spines, colonies and thicker cell walls), biochemistry (such as production of toxins, repellents) and in life history characteristics (formation of cysts, reduced recruitment rate). Our categorization of inducible defences in terms of the responsible induction mechanism provides guidance for future work, as hardly any of the available studies on marine or freshwater plankton have performed all the treatments that are required to pinpoint the actual cue(s) for induction. We discuss the ecology of inducible defences in marine and freshwater phytoplankton with a special focus on the mechanisms of induction, the types of defences, their costs and benefits, and their consequences at the community level.     

Radical sequestration by protein-bound 3,4-dihydroxyphenylalanine

Protein-bound 3,4-dihydroxyphenylalanine (PB-DOPA), a redox-active product of protein oxidation, is capable of functioning as both a pro- and antioxidant. A number of in vitro and in vivo studies have demonstrated a toxic, non-toxic or even beneficial effect of free DOPA, however little investigation has examined the physiological activity of PB-DOPA. Being the major treatment available for Parkinson's disease, most studies have focused on the effect of DOPA within neurological cells or tissues, although the presence of PB-DOPA in other locations, for example within atherosclerotic plaques, suggests that broader research is needed to fully understand the physiological effects of both free and PB-DOPA. We hypothesise that the generation of PB-DOPA can trigger an enhancement of the cellular antioxidant defence system, thus enabling PB-DOPA to restrict and potentially terminate the initiating oxidative stress, minimising the level of oxidative damage. Using luminol-enhanced chemiluminescence, we demonstrate that free DOPA is capable of direct peroxyl radical scavenging, even in the presence of competing scavengers, and has a different effect to that of the parent amino acid, tyrosine. Furthermore, we show that both free and PB-DOPA, in combination or individually, were able to protect monocytes and macrophages from peroxyl radical-induced oxidative stress in vitro. These results confirm a role for both free and PB-DOPA in cellular antioxidant defences and suggest the possibility of using DOPA as a potential therapeutic for the treatment of diseases involving oxidative stress or the accumulation of oxidative damage.     

Early herbivore alert matters: plant‐mediated effects of egg deposition on higher trophic levels benefit plant fitness

Induction of plant defences, specifically in response to herbivore attack, can save costs that would otherwise be needed to maintain defences even in the absence of herbivores. However, plants may suffer considerable damage during the time required to mount these defences against an attacker. This could be resolved if plants could respond to early cues, such as egg deposition, that reliably indicate future herbivory. We tested this hypothesis in a field experiment and found that egg deposition by the butterfly Pieris brassicae on black mustard (Brassica nigra) induced a plant response that negatively affected feeding caterpillars. The effect cascaded up to the third and fourth trophic levels (larval parasitoids and hyperparasitoids) by affecting the parasitisation rate and parasitoid performance. Overall, the defences induced by egg deposition had a positive effect on plant seed production and may therefore play an important role in the evolution of plant resistance to herbivores.     

Immunofluorescent detection of 8-oxoguanine DNA lesions in liver cells from aging OXYS rats, a strain prone to overproduction of free radicals

Production of free radicals in animals is accompanied with a number of pathologic conditions, some of which may be manifested through DNA damage. Studies of mechanisms of oxidative DNA damage by free radicals in vivo are hindered by the lack of good animal models with significant overgeneration of or increased sensitivity to free radicals. An inbred rat strain (OXYS) is characterized by inherited overgeneration of free radicals, lipid peroxidation, protein oxidation, DNA rearrangements, and pathological conditions paralleling several human degenerative diseases. We have used monoclonal antibodies against a common pre-mutagenic base lesion 8-oxoguanine (8-oxoG) in combination with indirect immunofluorescence microscopy and image analysis to follow the relative age-dependent amounts and distribution of 8-oxoG in liver cells from OXYS and Wistar rats. 8-OxoG increased with age in both strains of rats, with OXYS rats always displaying statistically significantly higher levels of oxidative DNA damage than Wistar rats. Statistical analysis indicates that 8-oxoG does not uniformly accumulate in all cells with advancing age or increasing free radical load, but rather concentrates in a minor fraction of cells with a high damage level.     

Interaction of reactive oxygen and nitrogen species with proteins

Free radicals and reactive oxygen or nitrogen species generated during oxidative stress and as by-products of normal cellular metabolism may damage all types of biological molecules. Proteins are major initial targets in cell. Reactions of a variety of free radicals and reactive oxygen and nitrogen species with proteins can lead to oxidative modifications of proteins such as protein hydroperoxides formation, hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, oxidation of sulfhydryl groups, oxidation of methionine residues, conversion of some amino acid residues into carbonyl groups, cleavage of the polypeptide chain and formation of cross-linking bonds. Such modifications of proteins leading to loss of their function (enzymatic activity), accumulation and inhibition of their degradation have been observed in several human diseases, aging, cell differentiation and apoptosis. Formation of specific protein oxidation products may be used as biomarkers of oxidative stress.     

α-Lipoic acid modulates thiol antioxidant defences and attenuates exercise-induced oxidative stress in standardbred trotters

Several micronutrient supplementation strategies are used to cope with oxidative stress, although their benefits have recently been questioned. The aim of the present study was to examine the effects of DL-α-lipoic acid (LA) in response to acute exercise and during recovery in horses. Six standardbred trotters were tested on the treadmill before and after 5-week LA supplementation (25 mg/kg body weight/day). According to electron paramagnetic resonance measurements, strenuous aerobic exercise increased significantly free radical formation in the gluteus medius muscle, which was prevented by LA supplementation. The activities of thioredoxin reductase and glutathione reductase in muscle were significantly increased in LA-treated horses, but neither LA nor exercise affected muscle thioredoxin activity. LA increased the concentration of total glutathione in muscle at rest and during recovery. Treatment with LA blunted the exercise-induced increase in plasma oxygen radical absorbance capacity and decreased the post-exercise levels of lipid hydroperoxides in plasma and malondialdehyde in plasma and in muscle. These findings suggest that LA enhances thiol antioxidant defences and decreases exercise-induced oxidative stress in skeletal muscle.     

Mitochondrial Changes in Ageing Caenorhabditis elegans – What Do We Learn from Superoxide Dismutase Knockouts?

One of the most popular damage accumulation theories of ageing is the mitochondrial free radical theory of ageing (mFRTA). The mFRTA proposes that ageing is due to the accumulation of unrepaired oxidative damage, in particular damage to mitochondrial DNA (mtDNA). Within the mFRTA, the "vicious cycle" theory further proposes that reactive oxygen species (ROS) promote mtDNA mutations, which then lead to a further increase in ROS production. Recently, data have been published on Caenorhabditis elegans mutants deficient in one or both forms of mitochondrial superoxide dismutase (SOD). Surprisingly, even double mutants, lacking both mitochondrial forms of SOD, show no reduction in lifespan. This has been interpreted as evidence against the mFRTA because it is assumed that these mutants suffer from significantly elevated oxidative damage to their mitochondria. Here, using a novel mtDNA damage assay in conjunction with related, well established damage and metabolic markers, we first investigate the age-dependent mitochondrial decline in a cohort of ageing wild-type nematodes, in particular testing the plausibility of the "vicious cycle" theory. We then apply the methods and insights gained from this investigation to a mutant strain for C. elegans that lacks both forms of mitochondrial SOD. While we show a clear age-dependent, linear increase in oxidative damage in WT nematodes, we find no evidence for autocatalytic damage amplification as proposed by the "vicious cycle" theory. Comparing the SOD mutants with wild-type animals, we further show that oxidative damage levels in the mtDNA of SOD mutants are not significantly different from those in wild-type animals, i.e. even the total loss of mitochondrial SOD did not significantly increase oxidative damage to mtDNA. Possible reasons for this unexpected result and some implications for the mFRTA are discussed.     

A short history of SFRR-Asia,its current status and significance

Antineoplastic agents are known to induce the production of free radicals leading to cell damage. These adverse effects may fuel the acquisition of new mutations and the development of treatment resistances. We selected 30 metastatic breast cancer patients receiving palliative chemotherapy, and paired blood samples, before and after chemotherapy, were extracted. We analysed DNA, lipid and protein oxidative damage markers and determined the extent of antioxidant and repair defences activation at the systemic level. We found that the DNA repair activity of the KU86 enzyme was significantly lower after chemotherapy and the antioxidant capacity of the plasma was significantly higher after treatment. Cox regression analysis revealed a significant effect of KU86 activity on the survival rates of those patients who received anthracyclines as part of their treatment. The high clinical heterogeneity of metastatic breast cancer patients warrants further studies to clarify the role of DNA repair and systemic antioxidant capacities during chemotherapy.     

Oxidative stress status in metastatic breast cancer patients receiving palliative chemotherapy and its impact on survival rates

Antineoplastic agents are known to induce the production of free radicals leading to cell damage. These adverse effects may fuel the acquisition of new mutations and the development of treatment resistances. We selected 30 metastatic breast cancer patients receiving palliative chemotherapy, and paired blood samples, before and after chemotherapy, were extracted. We analysed DNA, lipid and protein oxidative damage markers and determined the extent of antioxidant and repair defences activation at the systemic level. We found that the DNA repair activity of the KU86 enzyme was significantly lower after chemotherapy and the antioxidant capacity of the plasma was significantly higher after treatment. Cox regression analysis revealed a significant effect of KU86 activity on the survival rates of those patients who received anthracyclines as part of their treatment. The high clinical heterogeneity of metastatic breast cancer patients warrants further studies to clarify the role of DNA repair and systemic antioxidant capacities during chemotherapy.