Oxidative stress,antioxidants and stress tolerance

Traditionally, reactive oxygen intermediates (ROIs) were considered to be toxic by-products of aerobic metabolism, which were disposed of using antioxidants. However, in recent years, it has become apparent that plants actively produce ROIs as signaling molecules to control processes such as programmed cell death, abiotic stress responses, pathogen defense and systemic signaling. Recent advances including microarray studies and the development of mutants with altered ROI-scavenging mechanisms provide new insights into how the steady-state level of ROIs are controlled in cells. In addition, key steps of the signal transduction pathway that senses ROIs in plants have been identified. These raise several intriguing questions about the relationships between ROI signaling, ROI stress and the production and scavenging of ROIs in the different cellular compartments.     

Oxidative stress in mice

The aim of this study was to analyze the effect of high dietary Fe on liver antioxidant status in mice fed a corn-oil-enriched diet. Male Balb/c mice were fed for 3 wk with a standard diet enriched with 5% by weight of corn oil with adequate Fe (FCO diet) or supplemented with 1% carbonyl Fe (FCOFe diet). The control group was fed a standard diet. The high-Fe diet induced a twofold increase of hepatic Fe level. However, an increase of thymic Fe level has been induced solely by dietary fat. The hepatic copper (Cu) level slightly decreased in the FCO diet. In the spleen, the high-Fe diet-induced increase of Fe level was negatively correlated with the Cu level. The antioxidant status was influenced by both dietary fat and Fe. Mice fed corn-oil-enriched diets had a higher concentration of thiobarbituric acid-reactive substances (TBARS), with a greater increase in the FCOFe diet. Fatty acid analysis showed decreased n−3 and n−6/n−3 ratio, particularly in the FCOFe diet. Hepatic Cu/Zn superoxide dismutase (CuZn-SOD) activity was decreased in FCO diet, and Fe supplementation caused a further decrease in the enzyme activity. These results suggest that feeding with corn oil-enriched diet increases oxidative damage by decreasing antioxidant enzyme defense. The high-Fe diet additionally affects the antioxidant defense system, further increasing the tissue's susceptibility to lipid peroxidation. Additionally, both corn-oil- and Fe-enriched diets have increased the Cu requirement in mice.     

Oxidative stress,circulating antioxidants,and dietary preferences in songbirds

Oxidative stress is an unavoidable consequence of metabolism and increases during intensive exercise. This is especially problematic for migratory birds that metabolize fat to fuel long-distance flight. Birds can mitigate damage by increasing endogenous antioxidants (e.g. uric acid) or by consuming dietary antioxidants (e.g. tocopherol). During flight, birds may increase protein catabolism of lean tissue which may increase circulating uric acid and many birds also consume an antioxidant-rich frugivorous diet during autumn migration. We evaluated three related hypotheses in a migratory passerine: (1) protein consumption is positively related to circulating antioxidants, (2) a dietary oxidative stressor [i.e. polyunsaturated fatty acid (PUFA)] influences antioxidant capacity and oxidative damage, and (3) oxidative stress influences dietary antioxidant preferences. White-throated Sparrows (Zonotrichia albicollis) consuming a high protein diet increased circulating uric acid; however, uric acid, antioxidant capacity, and oxidative stress did not differ between birds consuming a high PUFA versus a low PUFA diet, despite increased oxidative damage in high PUFA birds. Birds did not prefer antioxidant-rich diets even when fed high PUFA, low protein. We conclude that White-throated Sparrows successfully mitigated oxidative damage associated with a high PUFA diet and mounted an endogenous antioxidant response independent of uric acid, other circulating antioxidants, and dietary antioxidants.     

Oxidative modification of citrate synthase by peroxyl radicals and protection with novel antioxidants

In mammals, aging is linked to a decline in the activity of citrate synthase (CS; E.C. 2.3.3.1), the first enzyme of the citric acid cycle. We used 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), a water-soluble generator of peroxyl and alkoxyl radicals, to investigate the susceptibility of CS to oxidative damage. Treatment of isolated mitochondria with AAPH for 8–24?h led to CS inactivation; however, the activity of aconitase, a mitochondrial enzyme routinely used as an oxidative stress marker, was unaffected. In addition to enzyme inactivation, AAPH treatment of purified CS resulted in dityrosine formation, increased protein surface hydrophobicity, and loss of tryptophan fluorescence. Propyl gallate, 1,8-naphthalenediol, 2,3-naphthalenediol, ascorbic acid, glutathione, and oxaloacetate protected CS from AAPH-mediated inactivation, with IC₅₀ values of 9, 14, 34, 37, 150, and 160?μM, respectively. Surprisingly, the antioxidant epigallocatechin gallate offered no protection against AAPH, but instead caused CS inactivation. Our results suggest that the current practice of using the enzymatic activity of CS as an index of mitochondrial abundance and the use of aconitase activity as an oxidative stress marker may be inappropriate, especially in oxidative stress-related studies, during which alkyl peroxyl and alkoxyl radicals can be generated.     

Oxidative stress is the master operator of drug and chemically-induced programmed and unprogrammed cell death: Implications of natural antioxidants in vivo

ROS, RNS, BRIs and ROS-RNS hybrids are produced during drug or chemical metabolism in vivo. These reactive species are instrumental to the culmination of cellular oxidative stress (OS). OS, once turned on, does not spare any vital intracellular macromolecule, such as glutathione, DNA, RNA, proteins, enzymes, lipids and ATP. Since concentration gradients of such components are very delicately balanced for normal cellular functioning, a gross perturbation leads to cell injury and cell death. Abundant evidence now suggests that intracellular antioxidants keep OS in check and maintain homeostasis. Our laboratory has focused on the role of OS in orchestrating various forms of cell death during drug and chemically-induced target organ toxicity and their counteraction by various natural or synthetic antioxidants in in vivo models. Despite complexity of the in vivo models, results show that metabolism of xenobiotics are invariably associated with different degrees of OS and natural antioxidants such as grape seed extract, bitter melon extract (Momordica charantia) and N-acetylcysteine (NAC) which were very effective in counteracting organ toxicities by minimizing events linked to OS (lipid peroxidation and total glutathione), and CAD-mediated DNA fragmentation. Phytoextract exposure rescued cells from toxic assaults, protected genomic integrity, and minimized apoptotic, necrotic and apocrotic (oncotic necrosis) cell deaths. Pre-exposure mode was more effective than post-exposure route. Overall scenario suggests that OS may have been the prime modulator of death and/or survival programs, whereas, antioxidants may have imparted a dual role in either erasing death signals or reviving survival signals, and a combination of antioxidants may be more beneficial than a single entity to influence a number of intracellular events operating simultaneously to neutralize chaotic toxicological consequences.     

Oxidative stress and antioxidants in tomato (Solanum lycopersicum) plants subjected to boron toxicity

BACKGROUND AND AIMS: Boron (B) toxicity triggers the formation of reactive oxygen species in plant tissues. However, there is still a lack of knowledge as to how B toxicity affects the plant antioxidant defence system. It has been suggested that ascorbate could be important against B stress, although existing information is limited in this respect. The objective of this study was to analyse how ascorbate and some other components of the antioxidant network respond to B toxicity. METHODS: Two tomato (Solanum lycopersicum) cultivars ('Kosaco' and 'Josefina') were subjected to 0.05 (control), 0.5 and 2 mm B. The following were studied in leaves: dry weight; relative leaf growth rate; total and free B; H(2)O(2); malondialdehyde; ascorbate; glutathione; sugars; total non-enzymatic antioxidant activity, and the activity of superoxide dismutase, catalase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase, ascorbate oxidase and l-galactose dehydrogenase. KEY RESULTS: The B-toxicity treatments diminished growth and boosted the amount of B, malondialdehyde and H(2)O(2) in the leaves of the two cultivars, these trends being more pronounced in 'Josefina' than in 'Kosaco'. B toxicity increased ascorbate concentration in both cultivars and increased glutathione only in 'Kosaco'. Activities of antioxidant- and ascorbate-metabolizing enzymes were also induced. CONCLUSIONS: High B concentration in the culture medium provokes oxidative damage in tomato leaves and induces a general increase in antioxidant enzyme activity. In particular, B toxicity increased ascorbate pool size. It also increased the activity of l-galactose dehydrogenase, an enzyme involved in ascorbate biosynthesis, and the activity of enzymes of the Halliwell-Asada cycle. This work therefore provides a starting point towards a better understanding of the role of ascorbate in the plant response against B stress.     

Oxidative stress and the use of antioxidants in diabetes: Linking basic science to clinical practice

Cardiovascular complications, characterized by endothelial dysfunction and accelerated atherosclerosis, are the leading cause of morbidity and mortality associated with diabetes. There is growing evidence that excess generation of highly reactive free radicals, largely due to hyperglycemia, causes oxidative stress, which further exacerbates the development and progression of diabetes and its complications. Overproduction and/or insufficient removal of these free radicals result in vascular dysfunction, damage to cellular proteins, membrane lipids and nucleic acids. Despite overwhelming evidence on the damaging consequences of oxidative stress and its role in experimental diabetes, large scale clinical trials with classic antioxidants failed to demonstrate any benefit for diabetic patients. As our understanding of the mechanisms of free radical generation evolves, it is becoming clear that rather than merely scavenging reactive radicals, a more comprehensive approach aimed at preventing the generation of these reactive species as well as scavenging may prove more beneficial. Therefore, new strategies with classic as well as new antioxidants should be implemented in the treatment of diabetes.     

Oxidative stress during aging of the yeast in a stationary culture and its attenuation by antioxidants

Oxidative stress during aging of Saccharomyces cerevisiae in stationary culture was documented by demonstration of progressive increase in the formation of superoxide, decrease in the content of acid‐soluble thiols and of acid‐soluble antioxidant capacity of cell extracts, and accumulation of aldehydes and protein carbonyl groups in two yeast strains and decreases in activities of antioxidant enzymes. Cells of a CuZn‐SOD (superoxide dismutase)‐1‐deficient strain showed a higher loss of viability than cells of an isogenic wild‐type strain. Cell survival was augmented, and changes in biochemical parameters were ameliorated, by addition of exogenous antioxidants (ascorbic acid, glutathione and melatonin) in both strains.     

Oxidative damage to catalase induced by peroxyl radicals: functional protection by melatonin and other antioxidants

Thermal decomposition by the azo initiator 2,2' azobis-(2-amidinopropane) dihydrochloride (AAPH) has been widely used as a water-soluble source of free radical initiators capable of inducing lipid peroxidation and protein damage. Here, in a lipid-free system, AAPH alone (40 mM) rapidly induced protein modification and inactivation of the enzyme catalase (EC 1.11.1.6). Using SDS-PAGE, it was shown that protein band intensity is dramatically reduced after 4 h of incubation with AAPH, leading to protein aggregation. Several antioxidants including melatonin, glutathione (GSH) and trolox prevented catalase modification when used at a 250 μM concentration whereas ascorbate was only effective at 1 mM concentration. All the antioxidants tested reduced carbonyl formation although melatonin was the most effective in this regard. Enzyme inactivation caused by AAPH was also significantly reduced by the antioxidants and again melatonin was more efficient than the other antioxidants used in this study. Results shown here demonstrate that alkyl peroxyl radicals inactivate catalase and reduce the effectiveness of cells to defend against free radical damage; the damage to catalase can be prevented by antioxidants, especially melatonin.     

Oxidative stress and antioxidant cell protection systems in the microaerophilic bacterium Spirillum winogradskii

The influence of oxygen availability during cultivation on the biosynthetic processes and enzymatic activities in the microaerophilic bacterium Spirillum winogradskii D-427 was studied, and the roles played by different systems of the defense against oxidation stress were determined. The metabolic adjustments caused by transition from microaerobic (2% O2) aerobic conditions (21% O2 of the gas phase) were found to slow down constructive metabolism and increase synthesis of exopolysaccharides as a means of external protection of cells from excess oxygen. This resulted in a twofold decline of the growth yield coefficient. Even though the low activity of catalase is compensated for by a multifold increase in the activities of other cytoplasmic enzymes protecting from toxic forms of O2--peroxidase and enzymes of the redox system of glutathione (glutathione peroxidase and glutathione reductase)--massive lysis of cells starts in the mid-exponential phase and leads to culture death in the stationary phase because of H2O2 accumulation in the periplasm (up to 10 micrograms/mg protein). The absence in cells of cytochrome-c-peroxidase, a periplasmic enzyme eliminating H2O2, was shown. It follows that the major cause of oxidative stress in cells is that active antioxidant defenses are located in the cytoplasm, whereas H2O2 accumulates in the periplasm due to the lack of cytochrome-c-peroxidase. The addition to the medium of thiosulfate promotes elimination of H2O2, stops cell lysis under aerobic conditions, lends stability to cultures, and results in a threefold increase in the growth yield.     

Oxidative stress in the developing brain: effects of postnatal glucocorticoid therapy and antioxidants in the rat

In premature infants, glucocorticoids ameliorate chronic lung disease, but have adverse effects on long-term neurological function. Glucocorticoid excess promotes free radical overproduction. We hypothesised that the adverse effects of postnatal glucocorticoid therapy on the developing brain are secondary to oxidative stress and that antioxidant treatment would diminish unwanted effects. Male rat pups received a clinically-relevant tapering course of dexamethasone (DEX; 0.5, 0.3, and 0.1 mg x kg(-1) x day(-1)), with or without antioxidant vitamins C and E (DEXCE; 200 mg x kg(-1) x day(-1) and 100 mg x kg(-1) x day(-1), respectively), on postnatal days 1-6 (P1-6). Controls received saline or saline with vitamins. At weaning, relative to controls, DEX decreased total brain volume (704.4±34.7 mm(3) vs. 564.0±20.0 mm(3)), the soma volume of neurons in the CA1 (1172.6±30.4 μm(3) vs. 1002.4±11.8 μm(3)) and in the dentate gyrus (525.9±27.2 μm(3) vs. 421.5±24.6 μm(3)) of the hippocampus, and induced oxidative stress in the cortex (protein expression: heat shock protein 70 [Hsp70]: +68%; 4-hydroxynonenal [4-HNE]: +118% and nitrotyrosine [NT]: +20%). Dexamethasone in combination with vitamins resulted in improvements in total brain volume (637.5±43.1 mm(3)), and soma volume of neurons in the CA1 (1157.5±42.4 μm(3)) and the dentate gyrus (536.1±27.2 μm(3)). Hsp70 protein expression was unaltered in the cortex (+9%), however, 4-HNE (+95%) and NT (+24%) protein expression remained upregulated. Treatment of neonates with vitamins alone induced oxidative stress in the cortex (Hsp70: +67%; 4-HNE: +73%; NT: +22%) and in the hippocampus (NT: +35%). Combined glucocorticoid and antioxidant therapy in premature infants may be safer for the developing brain than glucocorticoids alone in the treatment of chronic lung disease. However, antioxidant therapy in healthy offspring is not recommended.     

Oxidative stress protection by newly synthesized nitrogen compounds with pharmacological potential

In this study we used new nitrogen compounds obtained by organic synthesis whose structure predicted an antioxidant potential and then an eventual development as molecules of pharmacological interest in diseases involving oxidative stress. The compounds, identified as FMA4, FMA5, FMA7 and FMA8 differ in the presence of hydroxyl groups located in the C-3 and/or C-4 position of a phenolic unit, which is possibly responsible for their free radicals' buffering capacity. Data from the DPPH discoloration method confirm the high antiradical efficiency of the compounds. The results obtained with cellular models (L929 and PC12) show that they are not toxic and really protect from membrane lipid peroxidation induced by the ascorbate-iron oxidant pair. The level of protection correlates with the drug's lipophilic profile and is sometimes superior to trolox and equivalent to that observed for alpha-tocopherol. The compounds FMA4 and FMA7 present also a high protection from cell death evaluated in the presence of a staurosporine apoptotic stimulus. That protection results in a significant reduction of caspase-3 activity induced by staurosporine which by its turn seems to result from a protection observed in the membrane receptor pathway (caspase-8) together with a protection observed in the mitochondrial pathway (caspase-9). Taken together the results obtained with the new compounds, with linear chains, open up perspectives for their use as therapeutical agents, namely as antioxidants and protectors of apoptotic pathways. On the other hand the slight pro-oxidant profile obtained with the cyclic structures suggests a different therapeutic potential that is under current investigation.     

Oxidative stress in newborn infants with and without asphyxia as measured by plasma antioxidants and free fatty acids

A rapid perfusion of oxygen in infants at birth may cause an increase of oxidative stress. To assess this possibility, we measured levels of blood plasma antioxidants and free fatty acids in 20 normal infants at 0, 1, 3, and 5 days after birth. Plasma levels of the most reactive antioxidant, ascorbic acid, decreased daily to equilibrium values at days 3 and 5. Percentages of oxidized form of coenzyme Q-10 (%CoQ-10) in total coenzyme Q, another good marker of oxidative stress, in infants (25-31%) were significantly higher than those in healthy young adults (4.5%). Plasma levels of total free fatty acids (FFA) in normal infants were highest at day 1 and decreased rapidly thereafter. The content of polyunsaturated fatty acids (PUFA) in total FFA was lowest at day 1 and then increased. Since PUFA are susceptible to oxidation, these changes in FFA composition suggest that oxidative stress is most evident at the initial day of neonatal life. Furthermore, it appears that mono-unsaturated fatty acids such as oleic and palmitoleic acids increase in response to the oxidative loss of PUFA. Similar changes in plasma antioxidants, FFA levels, and FFA compositions were observed in 9 infants with asphyxia. Values of %CoQ-10 in infants with asphyxia were significantly greater than those in normal infants, suggesting that infants with asphyxia have elevated oxidative stress.     

Oxidative DNA damage,antioxidants, and cancer

Oxidised bases, such as 8-oxo-guanine, occur in cellular DNA as a result of attack by oxygen free radicals. The cancer-protective effect of vegetables and fruit is attributed to the ability of antioxidants in them to scavenge free radicals, preventing DNA damage and subsequent mutation. Antioxidant supplements (e.g., β-carotene, vitamin C) increase the resistance of lymphocytes to oxidative damage, and a negative correlation is seen between antioxidant concentrations in tissues and oxidised bases in DNA. Large-scale intervention trials with β-carotene have, however, led to increases in cancer. Recent measurements of the frequency of oxidised DNA bases indicate that earlier estimates were greatly exaggerated; there may be only a few thousand 8-oxo-guanines per cell. Convincing evidence for mutations resulting from oxidative damage, in tumours or cultured cells, is lacking. It seems that efficient antioxidant defences together with DNA repair maintain a steady-state level of damage representing minimal risk to cell or organism. BioEssays 21:238–246, 1999. © 1999 John Wiley & Sons, Inc.     

Oxidative stress, endogenous antioxidants, alcohol, and hepatitis C: pathogenic interactions and therapeutic considerations

Hepatitis C virus (HCV) is a blood-borne pathogen that was identified as an etiologic agent of non-A, non-B hepatitis in 1989. HCV is estimated to have infected at least 170 million people worldwide. The majority of patients infected with HCV do not clear the virus and become chronically infected, and chronic HCV infection increases the risk for hepatic steatosis, cirrhosis, and hepatocellular carcinoma. HCV induces oxidative/nitrosative stress from multiple sources, including inducible nitric oxide synthase, the mitochondrial electron transport chain, hepatocyte NAD(P)H oxidases, and inflammation, while decreasing glutathione. The cumulative oxidative burden is likely to promote both hepatic and extrahepatic conditions precipitated by HCV through a combination of local and more distal effects of reactive species, and clinical, animal, and in vitro studies strongly point to a role of oxidative/nitrosative stress in HCV-induced pathogenesis. Oxidative stress and hepatopathogenesis induced by HCV are exacerbated by even low doses of alcohol. Alcohol and reactive species may have other effects on hepatitis C patients such as modulation of the host immune system, viral replication, and positive selection of HCV sequence variants that contribute to antiviral resistance. This review summarizes the current understanding of redox interactions of HCV, outlining key experimental findings, directions for future research, and potential applications to therapy.