Oxidative Stress Spotlight
Oxygen metabolism, although essential for life, imposes a potential threat to cells because of the formation of partially reduced oxygen species.1,2 One electron reduction of oxygen produces superoxide whereas two electron reduction produces hydrogen peroxide. Therefore, electron flow through oxygen, utilizing processes such as the mitochondrial electron transport chain, flavoproteins, cytochrome P450 and oxidases, is tightly coupled to avoid partial reduction of oxygen.3
Normal cellular homeostasis is a delicate balance between the rate and magnitude of oxidant formation and the rate of oxidant elimination. Oxidative stress can, therefore, be defined as the pathogenic outcome of the overproduction of oxidants that overwhelms the cellular antioxidant capacity. Experimental support for oxidative stress as a mediator of cell death was provided recently by the finding that PC12 cells die following downregulation of Cu/Zn superoxide dismutase.4
Antioxidant defenses fall into two categories; enzymatic and nonenzymatic.1-3 Superoxide dismutases are metalloproteins that dismutate the superoxide radical (O2•) to hydrogen peroxide (H2O2) and molecular oxygen. Three types of superoxide dismutases are found in eukaryotic cells; Cu/Zn superoxide dismutase, predominantly located in the cytosolic fractions; Mn superoxide dismutase, located in the mitochondria, and EC superoxide dismutase, which is found in the extracellular space.1 Catalase, a heme protein located predominantly in peroxisomes and the inner mitochondrial membrane, catalyzes the conversion of H2O2 to H2O. In mammalian cells, the conversion of H2O2 to H2O is also accomplished by the reaction with glutathione catalyzed by glutathione peroxidases, a family of cytosolic selenoenzymes. Non-enzymatic defenses include small molecules such as membrane associated a-tocopherol, ascorbate and glutathione.
Biochemistry of Reactive Species: Free Radicals vs. Oxidants
The term free radicals has been equated with reactive species or oxidants. By definition, a radical is a molecule possessing an unpaired electron. Superoxide, nitric oxide, hydroxyl, alkoxyl and alkyl-peroxyl (lipid) are radicals. However, with the exception of hydroxyl radical none of these radicals are strong oxidants. Thus, not all radicals are strong oxidants and not all oxidants are radicals.
A critical function of reactive species is immunological host response. Generation of reactive species and strong oxidants by inflammatory cells is essential for killing invading microorganisms. However, experimental evidence has implicated reactive species in the pathogenic mechanism of several diseases. It is, therefore, important to understand the biochemical pathways for the induction of oxidative stress by reactive species. The most reasonable biochemical hypothesis is the reactive species-mediated modification of critical cellular targets.
Iron-sulfur enzymes are direct targets for superoxide and toxicity can be derived from the inactivation of these enzymes.1 Hydrogen peroxide at low mM levels does not react with many biological targets at an appreciable rate. However, the reaction of hydrogen peroxide with reduced divalent redox active metals such as iron can lead to the formation of strong oxidants. This reactivity of hydrogen peroxide may be important in biological oxidations of proteins and lipids that take place at the sites of metal binding. Divalent redox active metals can also catalyze the formation of the highly reactive hydroxyl by the metal-catalyzed Haber-Weiss reaction.5,6
However, hydroxyl radical reacts with almost all biological targets at rates exceeding 109 M-1sec-1 and therefore its diffusion distance inside a cell is minimal. Thus, in order for hydroxyl radical to cause toxicity it must be formed within a few Angstroms from a biological target.
An alternative pathway of superoxide toxicity is the formation of peroxynitrite by the reaction with nitric oxide.7 Nitric oxide is synthesized by nitric oxide synthases and mediates important physiological functions such as vasorelaxation, platelet aggregation, long term potentiation, and immune responses.8-11 The principal biological target of nitric oxide is guanylate cyclase and/or other iron-containing heme proteins. Nitric oxide is a radical but a weak one electron oxidant. Since both •NO and O2• are radicals they react rapidly to form peroxynitrite:
The second order rate constant of the reaction between nitric oxide and superoxide is 6.7 x 109 M-1sec-1 which is nearly three times faster than the reaction of superoxide with superoxide dismutase (2.9 x 109 M-1sec-1) and nearly thirty times faster than the reaction of •NO with heme proteins. This implies that the formation of peroxynitrite can out-compete the major scavenging pathways for •NO and O2•. Peroxynitrite is not a free radical but a strong one or two electron oxidant and nitrating agent.12-15 Although peroxynitrite can oxidize most biological molecules similar to the hydroxyl radical, the rate constants of the biological oxidations of peroxynitrite are 10,000 fold slower than the rate of hydroxyl radical. This implies that peroxynitrite will diffuse much further than the hydroxyl radical and will react with selective targets. The targets are determined for the most part by the rate by which they react with peroxynitrite. The fastest reactions for peroxynitrite presently are the reactions with Zn-S and Fe-S centers with metalloproteins and carbon dioxide.12,15 Whereas the Zn-S and Fe-S centers will be oxidized, the last two reactivities will promote nitration of tyrosine residues on proteins. Protein nitrotyrosine has been detected in human diseases and experimental models of disease that do not involve an inflammatory process.7
Cellular Responses to Reactive Species: Time and Magnitude of Exposure
The flux and the time of exposure are critical factors in determining the outcome of oxidative stress. Aging can be considered the result of a continuous exposure to a low flux of reactive species over the life span. Although the antioxidant networks maintain the critical balance towards physiology, a few reactive species escape the surveillance of the antioxidant network and react with biological targets. Oxidation of biological targets will not necessarily translate to expression of a phenotype because repair processes may sustain normal physiologic function. However, as the frequency of oxidation of biological targets increases (and possibly as repair processes slow), detection of oxidized proteins, lipids and even DNA becomes apparent with aging and other reactive-species mediated pathologies.16-18
Severe oxidative stress results in necrotic cell death. Generation of reactive species during hyperoxia (breathing of >95% oxygen) or reperfusion of an ischemic tissue leads to tissue necrosis.19-20 A moderate exposure to reactive species can also result in cell death that usually occurs 20-24 hours after the initial insult. In most cases delayed cell death resembles apoptosis since DNA fragmentation and other features of apoptosis are evident. It is not clear how reactive species can induce delayed cell death or apoptosis. Potential pathways that once altered by reactive species will lead to delayed cell death include energy sources (mitochondria, activation of Poly- ADP ribosyl synthase), ionic homeostasis, signal transduction and membrane structural integrity.4,21,22
Overall, the inherent ability of cells to withstand oxidative stress is dependent upon several factors: their antioxidant capacity, the ability to sustain metabolic requirements by deriving energy from alternate pathways, efficiency to repair oxidatively modified biomolecules, and availability and utilization of trophic support.
Reactive Species and Signal Transduction
Recently, evidence has suggested that reactive species can be utilized in signal transduction events.23-27 Signal transduction for the most part is viewed either as a specific interaction between proteins or events mediated by second messenger molecules such as Ca2+ and cyclic nucleotides. Nitric oxide can be clearly considered a signal transducing molecule because it specifically activates guanylate cyclase. However, except for nitric oxide, specific targets that can be utilized in signal transduction are not known for other reactive species. Moreover, the steady state levels of reactive species such as superoxide and hydrogen peroxide are under the control of enzymatic pathways. For example, the steady state levels of superoxide in superoxide dismutase deficient E. coli is 5 x 10-7M (taking into account scavenging by glutathione) whereas in superoxide dismutase proficient E. coli the levels are 2 x 10-10 M.28 Therefore, the lack of specificity and the low intracellular levels creates difficulty in explaining how reactive species can be utilized in signal transduction.
The answer to this question in part can be found in the biochemical reactivities and the cellular targets for reactive species. Superoxide, nitric oxide and peroxynitrite react with Fe-S and Zn-S centers. Fe-S and Zn-S centers are not only found in enzymes regulating bioenergetics but also in transcription factors and in iron regulatory proteins. Peroxynitrite is also a nitrating agent that nitrates tyrosine residues in proteins. Nitration alters the pKa of tyrosine residues and interferes with the ability of tyrosine kinases to phosphorylate.29,30 The activity of different kinases, transcription factors and ion channels is redox sensitive and is dependent on a critical cysteine residue which can be modified by reactive species. Finally, reactive species can indirectly induce signal transduction events by inducing mitochondrial Ca2+ release and lipid peroxidation. These signaling pathways may be critical in mediating apoptosis or delayed cell death.
How to Detect and Quantify Reactive Species
The short half life of most reactive species in biological systems does not permit for their direct detection and quantification.3,5,6,23 Therefore, detection of reactive species relies on indirect measurement of modified targets. If you will, consider reactive species as sharks. Their presence in biological systems is therefore determined by the “bite marks” formed on critical cellular targets. In simple in vitro assays, the task of detection and quantification of reactive species is relatively well established. However, as one moves from the simple test tube assay, to cells in culture, to isolated organs, to whole animals or humans, the difficulty in detecting these “bite marks” increases exponentially. The ability to detect and quantify reactive species is a function of the amount of modified molecules present at a given time and the sensitivity of the assay. Biological targets that have been utilized for detection of oxidative modification include lipids, proteins, thiols and DNA. Reactive species react with more than one biological target and since the concentration of biological targets varies among cells, it is difficult to predict which target will be preferentially modified. Therefore, in more complex systems, it may be necessary to measure more than one end-point modification of biological targets. For example, measurement of the reduced to oxidized glutathione ratio will reflect a degree of oxidative stress but will not be useful in elucidating potential pathways responsible for the oxidation. In some models interference with the formation of the potential reactive species maybe useful in elucidating the reactive pathways.
Another method for detecting reactive species is the use of “reporter” compounds that are oxidized by reactive species to either chromogenic, fluorescent, luminescent or Electron Paramagnetic Resonance products. These probes have been utilized in cells, isolated organs and whole animal models and fall in two categories: cell permeable and non-permeable compounds. Intracellular detection requires a substrate that has a reasonably fast rate of reaction with reactive species and can be delivered at high enough concentrations to out-compete antioxidant and scavenging pathways. Extracellular detection represents the fraction of reactive species that are either generated very close to cell membrane or escape the antioxidant and scavenging networks and have not been reacted with cellular targets. This implies that the magnitude of the stress inside the cell could be significantly higher compared to what is measured extracellularly.
My apologies to all the scientists that over the years have made significant contributions to this field but their work is not cited here due to space limitations.
1. Fridovich, I., 1986. Arch. Biochem. Biophys. 247, 1.
2. McCord, J.M., and Fridovich, I. 1988. Free Rad. Biol. Med. 5, 363.
3. Freeman, B.A., and Crapo, J.D. 1982. Lab. Invest. 47, 412.
4. Troy, C.M., et al. 1996. J. Neurosci. 16, 253.
5. Buettner, G.R. 1993. Arch. Biochem. Biophys. 300, 535.
6. Halliwell, B., and Gutteridge, J.M.C. 1990. Trends Biochem. Sci. 15, 129.
7. Beckman, J. 1996. J. Chem. Res. Toxic. 9, 836.
8. Furchgott, R.F. 1996. JAMA 276, 1186.
9. Murad, F. 1996. JAMA 276, 1189.
10. Ignarro, L.J., et al. 1990. Annu. Rev. Pharmacol. Tox. 30, 535.
11. Moncada, S., et al. 1991. Pharmacol. Rev. 43, 109.
12. Koppenol, W.H., et al. 1992. Chem. Res. Toxicol. 5, 834.
13. Radi, R., et al. 1991. J. Biol. Chem. 266, 4244.
14. Crow, J.P., et al. 1995. Biochemistry 34, 3544.
15. Ischiropoulos, H., et al. 1992. Arch. Biochem. Biophys. 298, 431.
16. Ames, B.N., et al. 1995. Biochim. Biophys. Acta 127, 165.
17. Stadtman, E.R. 1992. Science 257, 1220.
18. Imlay, J.A., and Linn, S. 1992. Science 240, 1302.
19. Turrens, J.F., et al. 1982. Arch. Biochem. Biophys. 217, 411.
20. McCord, J.M.N. 1985. Engl. J. Med. 312, 159.
21. Dawson, V.L., and Dawson, T.M. 1995. Adv. Pharmacol. 34, 323.
22. Szabo, C., et al. 1996. Proc. Natl. Acad. Sci. USA 93, 1753.
23. Lander, H.M. 1997. FASEB J. 11, 118.
24. Suzuki, Y.J., et al. 1997. Free Rad. Biol. Med. 22, 269.
25. Meyer, M., et al. 1993 EMBO J. 12, 2005.
26. Pantopoulos, K., et al. 1994. Trends Cell Biol. 4, 82.
27. McConkey, D.J., and Orrenius, S. 1994. Trends Cell Biol. 4, 370.
28. Imlay, J.A., and Fridovich, I.J. 1991. J. Biol. Chem. 266, 6957.
29. Gow, A., et al. 1996. FEBS Lett. 385, 63.
30. Chance, B., and Gao, G. 1994. Environ. Health Persp. 10, 29.
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