Reviews of Articles on Oxidative Stress

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Reactive Oxygen Species in Health and Disease. Alfadda, A.A. and Sallam, R.M. (2012) J.Biomed.Biotechnol. 2012, 936486.
Generally speaking, a state of homeostasis means a state of good health. The higher organisms, including mammals have mechanisms in place to ensure free radicals (of which reactive oxygen species are the most prevalent) homeostasis. In the early stages of free radical research the focus was on the relationship between ROS activity and the conditions they help create such as cardiovascular diseases, cancer and metabolic disorders, i.e. diabetes, chronic inflammation and insulin resistance. Later it became apparent that living organisms cannot do without ROS involvement in processes like cell signaling, vascular tone or defense against bacterial/viral infection and inhibition of tumor growth.
The present review has the merit of looking at both sides of the ROS coin, the good and the bad. Thus, the authors provide several examples of ROS role in normal physiological processes such as normal vascular diameter regulation, oxygen sensing, immune system activity, skeletal muscle physiology, signal transduction and genomic stability. Attention is also paid to the mechanisms by which two cellular organelles, namely mitochondria and endoplasmic reticulum help maintain cellular homeostasis. A fair amount of space is devoted to the role of ROS in metabolic disorders, chronic inflammation, mitochondrial dysfunction as well as their role in obesity-associated pathologies. Finally, emphasis is placed on antioxidant therapies and how these approaches can help medical practice.
In our opinion, this review article is very well written, not too long, not too short, the exact length to keep readers "hooked" until the end. It provides a good source of information for those already familiar with the subject as well as for the newcomers. Highly recommended.

  Radical-free biology of oxidative stress. Jones, D.P. (2008) Am.J.Physiol. Cell Physiol. 295(4) C849-C868.
"Not all free radicals need be harmful". This is a statement that acknowledges a shift in the free radical paradigm of oxidative stress that seems to be taking hold in the thinking of the biological oxidation scientific community. It has become increasingly clear in recent years that free radicals such as NO. and the nonradical oxidant H2O2 at low concentrations are major players in cell signaling and physiological regulation.
Although in simple terms a postulate means "something taken to be true without proof" the four postulates, as laid down by Dr. Jones in his excellent review article: 'Radical-free biology of oxidative stress', are in fact supported by a large body of evidence and therefore can no longer be considered as postulates in the proper sense of the word. Because of the importance of the subject for the future development of oxidative stress research and to give readers an insight into the "redox hypothesis", as it is called by the author we reproduce the four postulates as laid down in the article:
1. All biological systems contain redox elements (e.g. redox-sensitive cysteine residues) that function in cell signaling, macromolecular trafficking and physiological regulation; 2. Organization and coordination of the redox activity of these elements occurs through redox circuits dependent on common control nodes (GSH, thioredoxin); 3. The redox-sensitive elements are spacially and kinetically insulated so that "gated" redox circuits can be activated by translocation/aggregation and/or catalytic mechanisms; 4. Oxidative stress is a disruption of the function of these redox circuits caused by specific reactions with the redox-sensitive thiol elements, altered pathways of electron transfer or interruption of the gating mechanisms controlling the flux through these pathways.
An important point (supported by data from numerous investigators) raised by this hypothesis is that cysteine (Cys) residues in many proteins involved in signaling and control undergo reversible oxidation reactions. This is in sharp contrast with the classical view according to which the thiol groups are normally fully reduced and the role of GSH and other reducing agents is to protect against oxidation by reactive oxygen species (ROS). In fact, the existing data indicate that a relatively rapid continuous oxidation of thiols occurs. It was estimated that thiol oxidation takes place at a rate of 0.5% of the cellular thiol content per minute. There is increasing evidence that critical Cys residues, which are involved in redox signaling and control are not in equilibrium with each other or the NADPH/NADP+ couple. These Cys residues appear to act as sulfur switches according to an "on-off" type mechanism and are controlled by redox nodes represented by the main redox couples, i.e. GSH/GSSG, thioredoxinred/thioredoxinox and cysteine/cystine (Cys/CySS). These redox couples, which act as control points in a network are maintained under stable, nonequilibrium conditions in biological systems. Pathological conditions that are linked to an increased oxidative stress may not be the consequence of high free radical activity caused by ROS such as HO. and O2- but rather of a disruption of the function of redox circuits.
The article presents convincing evidence from many studies that strengthen the assertions made by the four postulates. In addition, there is good reason to believe that H2O2 acts as a second messenger for a number of signal transduction pathways. Hypothetical models are also put forth that could help investigators develop redox systems models that will better characterize signaling pathways with multiple redox sensitive steps. Since many disease states are associated with changes in redox states and/or regulation of protein thiol modification the development of a comprehensive systems biology theory that includes redox systems biology is critical for understanding how redox networks function and how they are integrated and controlled at cellular level. This is of paramount importance because family physicians and the public at large are often confused as to what level of dietary antioxidants is optimal for maintaining good health especially when faced with messages from aggresive marketers promoting mega doses of antioxidant vitamins. Is more better? Apparently not because mega doses of the usual ROS scavenging vitamins may do more harm than good by disrupting the normal redox circuitry. Moreover, several large scale epidemiological studies testing the usefulness of high antioxidant vitamin supplementation failed to show clear clinical benefits.
In conclusion, this well written review article is a must read for well established investigators as well as newcomers to the field of oxidative stress research. We can only but look forward to new developments in this exciting area of biomedical research.

NADPH oxidases, reactive oxygen species and hypertension. Clinical implications and therapeutic possibilities. Paravicini, T.M. & Touyz, R.M. (2008) Diabetes Care 31, S170-S180.
There is compelling evidence from animal experimental models that oxidative stress OS) is positively linked to hypertension (HT). Human studies did not yield as convincing results as the animal studies but enough evidence has accumulated to show that OS is clearly increased in patients with essential, renovascular, salt-sensitive, cyclosporine-induced HT and preeclampsia. Polymorphonuclear leukocyte-and platelet-derived superoxide was significantly increased in hypertensive patients, which would suggest a systemic OS in these patients. Besides these circulatory cells, superoxide is generated by all vascular cell type such as endothelial, smooth muscle and adventitial cells. The enzyme sources of interest in vascular disease and HT are: xanthine oxidase, uncoupled nitric oxide synthase and NAD(P)H oxidase. This review article focuses on the latter enzyme, which occurs as a multi-subunit complex. They are expressed in many tissues and mediate diverse biological functions. There is still a lot to be learned about how NADPH oxidase subunits interact in vascular cells and how they generate superoxide. Besides their role in normal cellular processes ROS have been associated with pathological events like endothelial dysfunction, smooth muscle cells proliferation and extracellular matrix deposition. Whether the increased ROS activity in cardiovascular disease is the primary cause of these conditions or simply associated with it is a question that has remained largely unanswered. Strategies for reducing the OS either through controlling NADPH oxidase activity or antioxidant supplementation are also discussed in this article. It is hoped that by better understanding the mechanisms regulating NADPH oxidase activity, therapies can be devised to reduce the damaging effects of ROS. It is also important to develop sensitive and specific biomarkers to assess the OS status in HT patients, especially at the onset of the condition so that effective treatment can be devised to prevent HT to evolve to cardiovascular disease.

The role of mitochondrial DNA mutations in aging and sarcopenia: Implications for the mitochondrial vicious cycle theory of aging. A. Hiona & C. Leeuwenburgh, (2008) Exp.Gerontol. 43(1) 24-33.
It is beyond dispute that ROS can cause considerable damage to mitochondrial DNA (mtDNA), which results in mutations and ultimately in mitochondrial decay and tissue dysfunction. Mutations can also arise from sporadic errors during mtDNA replication. The main themes of this minireview deal with: 1) the possible role of mtDNA mutations in aging-associated phenotypes such as muscle loss (sarcopenia) and 2) the involvement of ROS in mtDNA mutagenesis and other processes leading to mitochondrial decay, apoptosis and tissue dysfunction. There is compeling evidence to suggest that mtDNA mutations/deletions are a contributing factor in mammalian aging. Thus, it was found that a specific mutation in the exonuclease domain of DNA polymerase g (PolyG) led to an increased spontaneous mutation rate in mtDNA in mice homozygous for the mutation. This increase in somatic mtDNA mutations was associated with reduced lifespan and premature onset of aging-related phenotypes such as osteoporosis, heart enlargement and sarcopenia. At molecular level, heart and liver tissues exhibited no increased DNA oxidation and protein carbonylation. Interestingly, in the PolyG mice, the accumulation of mtDNA mutations was not associated with an increase of oxidative stress or defects in cellular proliferation. In the wild type, most of superoxide is generated by the complex I and III of the electron transport chain (ETC) so mtDNA is subject to intense superoxide attack due to its proximity to the site of free radical generation. Not long ago the original theory of aging put forward by Harman in the 1950s got a new boost by the so-called 'vicious cycle' theory of aging. According to this theory, accumulated mtDNA mutations lead to increased ROS production, which in turn cause more mutations in the mtDNA and this will exponentially increase damage and dysfunction. However, this theory has been recently challenged by evidence showing that mutations, which affected the synthesis of cytochrome b did not result in higher ROS production. In addition, PolyG-deficient mice exhibited an increase in cytosolic mono- and oligonucleosomes, which is indicative of apoptosis. At present, there is no conclusive evidence arguing for the predominance of either of the two mechanisms. It is likely that each one has its own contribution to the aging process through mitochondrial dysfunction followed by apoptosis and tissue function loss. Aging is a complex process and is likely to have multi-factorial causes. Until a clearer picture of the cause and role of mtDNA mutations in aging emerges this area of research will continue to be hotly debated.

  Inhalation of hydrogen gas suppresses hepatic injury caused by ischemia/reperfusion through reducing oxidative stress. Fukuda, K. et al. (2007) Biochem.Biophys.Res.Commun. 361(3) 670-674 and Hydrogen acts as a therapeutic antioxidant by selectively reducing cytoxic pxygen radicals. Ohsawa, I. et al. (2007) Nat.Med. 13(6) 688-694.
A new antioxidant in the fight against ROS seems to make its entrance on the oxidative stress stage. Two recent papers by Japanese researchers reported that molecular hydrogen can significantly reduce the oxidative stress in animal models. Thus, by inhaling molecular hydrogen rats that had been oxidatively stress by ischemia/reperfusion in hepatic and brain tissues showed a significant reduction of tissue damage. While hydrogen gas appears effective against HO. (the most toxic of all ROS) it did not scavenge other ROS, i.e. superoxide and hydrogen peroxide. If the results are confirmed by other research groups hydrogen therapy may open a new way for tackling the in vivo deleterious action of ROS, particularly in conditions such as heart stroke when ischemia/reperfusion cause a lot of tissue damage because of free radicals attack.

Free radicals and antioxidants in normal physiological functions and human disease. Valko, M. et al. (2007) Int.J.Biochem. Cell Biol. 39(1) 44-84.
For those who are not very familiar with the "two-faced" character of free radicals, particularly those of oxygen (ROS) and nitrogen (RNS) here is a review article that will give them the insight into these fascinating chemical species. Besides being toxic for cells at higher concentration, ROS/RNS are now known to have beneficial effects such as defence against infectious agents, involvement in several signalling pathways and the induction of a mitogenic response. Depending on their intracellular concentration the ROS can tip the redox balance (redox homostasis) of cells toward a more oxidant environment (higher concentration) or toward a more reducing environment (lower concentration). The reader of this review will learn about the biochemistry of free radicals and how they are generated, the damage caused by ROS to cell's biomolecules, the role of antioxidants in maintaining the redox homeostasis, the role of ROS/RNS in redox regulation of normal physiological processes and the pathophysiological implication of altered redox regulation such as it occurs in several diseases and aging. The attention paid to free radical research is warranted by their involvement in the pathogenesis of cardiovascular disease, neurodegenerative diseases, autoimmune disorders, cancer and possibly others. This review article will interest postgraduate students in life sciences, biomedical researchers and nutritionally-oriented physicians who may wish to learn more about the involvement of free radicals in human pathology and what can antioxidant-rich foods do to help maintain the redox homeostasis, which is crucial for the good health of cells/tissues and hence the whole body.

No Evidence for Oxidative Stress as a Mechanism of Action of Hyperhomocysteinemia in Humans. Huerta, J.M. et al., Free Radical Research 11, 1215-1221 (2004).
It took a long time for the hypothesis put forward some 30 years ago by K.S. McCully regarding the involvement of homocysteine in the pathophysiology of atherosclerosis to get general acceptance. Thus, there is compelling evidence to suggest that elevated plasma homocysteine is a predicator of cardiovascular disease (CVD), independently of other known risk factors although the mechanism involved remains largely undefined.
Previous studies indicated that homocysteine is susceptible to autooxidation with subsequent generation of reactive oxygen species (ROS). Some of these ROS such as superoxide can react with NO. to form HOONO thus lowering the level of NO., which plays an important role in vasodilator and anticoagulant function of the endothelial cells. It was also found that administration of vitamins C and E alleviated the adverse effects of hyperhomocysteinemia on the endothelial cell function. All these findings and others lent support to the idea that an elevated level of homocysteine in blood plasma could be a risk factor in developing heart disease and the mechanism involved has an oxidative stress component.
The work by Huerta et al. provides evidence that an elevated level of plasma homocysteine in a cohort of healthy elderly of both gender is not associated with oxidative damage as indicated by the level of lipid peroxidation as well as the antioxidant status in blood plasma. Thus, the plasma malone dialdehyde concentration (a marker of lipid peroxidation, hence oxidative stress) does not increase as the level of homocysteine increases. Moreover, there was no positive correlation between hyperhomocysteinemia and the activity of two antioxidant enzymes (superoxide dismutase and glutathione peroxidase) and the plasma level of nonenzymatic antioxidants such as vitamin E, b-carotene, retinol. However, by the authors' admission their data should not be seen as conclusive since the work was carried out on a small cohort of healthy individuals, with normal to moderately higher homocysteine levels so the results cannot be extrapolated as to predict how high plasma homocysteine would affect lipid peroxidation. The results of this study would suggest that oxidative stress and hyperhomocysteinemia act as independent risk factors for CVD.

Tryptophan Loading Induces Oxidative Stress. C.M. Forrest et al., Free Radical Research 38(11), 1167-1171 (2004).
This paper provides evidence that tryptophan loading to healthy volunteers resulted in increased lipid peroxidation (as determined by the measurement in the blood plasma of two end products of lipid peroxidation, i.e. 4-hydroxynonenal and malone dialdehyde) after 5 and 7 hours following oral administration of the amino acid. Tryptophan loading also activates the oxidative pathway of tryptophan degradation. One of the intermediate metabolites in the degradation pathway is 3-hydroxykynurenine, which was shown in a previous study to induce neuronal cell death through the accumulation of H2O2. The oxidative stress caused by the reactive oxygen species such as, H2O2 and possibly O2- lead to cell death in the brain tissue.
An important conclusion of this study is that caution should be exercised when tryptophan is administred to patients with psychiatric disorders, e.g. depression, because of the potential damaging effects caused by an increased oxidative stress, particularly in the brain. In addition, the results of the present study suggest that dietary regimens rich in tryptophan such as the Atkins diet, may also in the long run, create conditions for the development of oxidative stress.

Evaluation of a Multi-parameter Biomarker Set for Oxidative Damage in Man: Increased Urinary Excretion of Lipid, Protein and DNA Oxidation Products after One Hour of Exercise.
Orhan, H. et al., Free Radical Research 38(18), 1269-1279 (2004).
One important challenge faced by the biomedical scientists working on the interaction of reactive oxygen species (ROS) with biological systems is finding the biomarkers that can best describe the degree of oxidative stress an organism is subjected to. In humans, the focus is on how well these biomarkers of oxidative stress can be used as a predictor of developing an oxidative stress-related disease. Athlets are an excellent study group because exercising appears to oxidatively damage muscle tissue and possibly other organs. The long used malone dialdehyde (MDA) test to assess lipid peroxidation in blood plasma yielded conflicting results when athlets were tested because of the large inter-individual variability and the non-specificity of the methods for determining MDA level in a particular biological sample.

In the present study Orhan and al. attempted to evaluate a set of biomarkers that could be used to monitor oxidative damage in humans. This set of biomarkers targeted lipids, proteins and DNA for oxidative damage. All biomarkers were analyzed in urine samples obtained after exercising for one hour. Several aldehydes, o, o'-dityrosine and 8-hydroxy deoxyguanosine (8-OHdG) were the biomarkers for lipid, protein and DNA oxidation, respectively. Among the various aldehydes tested urinary butanal appears to be more reliable marker for lipid oxidative damage than MDA or acetone. The latter is also formed through ketosis (the degradation of free fatty acids to ketone bodies), so it does not come exclusively from products of lipid peroxidation. Urinary MDA level was not increased after moderate exercising, which is in agreement with the fact that urine is not the major route for MDA excretion. So, even if MDA was produced as a result of lipid oxidation it does not show in urine samples at higher than normal levels. o, o'-dityrosine was found to be a reliable, sensitive and early biomarker of protein oxidative damage. 8-OHdG appears to be a good biomarker for DNA oxidative stress but caution should be execised when this marker is assayed by the ELISA technique, in the sense that the values obtained by this technique are meaningful only when relative urinary 8-OHdG levels are compared and not its absolute concentration in the biological sample. My opinion is that the present paper is a neat piece of work that has the merit of putting forward the first application of a large, multiple biomarker set for assessing lipid, protein and DNA oxidative damage after physical effort. Hopefully, in not so distant future such biomarkers will become common practice for assessing oxidative damage in large cohorts similar to those used today for checking liver and heart health status as well as other body functions.

Meta-Analysis: High-Dosage Vitamin E Supplementation May Increase All-Cause Mortality. E.R. Miller et al., Annals of Internal Medicine 142(1) 37-46 (2005).
According to this review article vitamin E supplementation at doses above 400 IU/day does not get good marks and is to be avoided, particularly in populations at high risk for a chronic disease. The authors analyzed 19 clinical trials of which some 50% used vitamin E alone while the rest combined vitamin E with other vitamins and minerals. In order to ensure a standardization across the surveyed studies the authors converted the vitamin E dosages reported in the studies being analyzed to IU/day relative to all rac- a-tocopherol acetate. However, it is not clear from this study if any distinction was made between the natural and synthetic forms of vitamin E since it has been shown that there are differences in the bioavailability between the two forms.
Unfortunately, the publication of this article first on the journal's website was announced with a lot of hype in several newspapers around the world with headlines such as "Lethal Consequences of Vitamin E Overdose", High Doses of Vitamin E Deadly", etc. This kind of coverage does more harm than service to the practice of keeping the general public well informed about the progress in the biomedical sciences, the more so as the pooled all-cause mortality risk difference in high-dosage vitamin E trials was 39 per 10,000, which really does not amount to something that would warrant the afore mentioned headlines. If it was not for the headlines, the present article may have never reached the national newspapers and would have ended as yet another study in the pile of published reports dealing with a long disputed subject: are the antioxidant nutritional supplements a real help in the prevention of a host of degenerative diseases? But because of the headlines several members of the science community including researchers, biomedical writers and medical practitioners "bothered" to make their comments known, as rapid response letters posted on the journal's website that you can read here.

Antioxidant vitamins and coronary heart disease risk: a pooled analysis of 9 cohorts. Paul Knekt et al., Am.J.Clin.Nutr. 80, 1508-1520 (2004).
To date we still don't have a conclusive answer as to the amount of antioxidant nutrients such as the vitamins Ca and E, carotenes and selenium from fresh fruits and vegetables, not to mention from nutritional supplements in pill form, one should take on a daily basis in order to significantly lower the risk of coronary heart disease (CHD). Previous cohort studies on the subject failed to yield consistent results due to a variety of factors such as a lack of power to detect associations between the intake of various antioxidant factors and the reduced risk of CHD, unsatisfactory control for potential confounding variables, an inability to investigate subpopulations, etc. In addition, randomized intervention trials in primary intervention of CHD have not shown significant benefits from vitamin E and a-carotene supplementation.

In the present study Knekt and his associates set out to analyze pooled data from 9 major cohort studies dealing with diet and CHD. The large database available to the authors enabled them to tackle issues such as whether single antioxidants or in combination could better predict the occurrence of CHD, or whether nondietary or dietary risk factors of CHD can alter the association. By using a variety of criteria for assessing the relationship between the dietary and nondietary antioxidants and the likelyhood of developing CHD as well as several statistical methods to analyze the data the authors concluded that the use of vitamin C supplementation may reduce CHD incidence in both genders. There was only a slight chance that vitamin E and carotene lutein may be beneficial. Besides some advantages afforded by the pooled data from these cohort studies such as the ability to differentiate the associations for dietary and supplemental intakes and a more satisfactory control of confounding factors there were still some methodological factors that may have masked associations or caused artificial associations. For example, there is a possibility that other carotenoids or single vitamin E compounds or a combination of them may have been responsible for the observed protection. Also, changes in dietary habits during the follow-up period in some cohorts may have biased the observed associations between antioxidant intake and CHD. It is also apparent that because of the metabolic interplay between the various antioxidants the inhibition of LDL oxidation (the marker most commonly used as a measure of oxidative stress) by several antioxidants cannot be ruled out.

As the conflicting results of numerous epidemiologic studies have shown, to rely on higher antioxidant intakes, whether from diet (fruit and vegetables) or non-diet supplementation is not the only way to go. Until the effects of higher antioxidant intake, particularly vitamins C and E are fully understood it appears, from the present study that higher doses of antioxidant vitamins such as vitamin C cannot be recommended solely as a means to lower the risk of developing heart disease. Because of the complexity of the pathological itself, which involves genetic as well as metabolic factors, coupled with dietary and lifestyle habits, finding "the magic bullet" for the prevention of CHD seems as elusive as ever.

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