key: cord-017188-d3xg05ty
authors: Swartz, H.M.; Mason, R. P.; Hogg, N.; Kalyanaraman, B.; Sarna, T.; Plonka, P.M.; Zareb, M.; Gutierrez, P. L.; Berliner, L. J.
title: Free Radicals and Medicine
date: 2005
journal: Biomedical EPR, Part A: Free Radicals, Metals, Medicine, and Physiology
DOI: 10.1007/0-387-26741-7_3
sha: 
doc_id: 17188
cord_uid: d3xg05ty

EPR has been employed in attempts to understand the basis of specific pathophysiologies in which free radicals have a postulated role. Examples described include pulmonary free radical damage, free radicals and sickle cell disease, free radicals in amyotrophic lateral sclerosis, melanin and free radicals and the potential role of oxidative stress in the induction of cancer. The final section of the chapter describes the use of NMR as the spectroscopic measure of spin-trapped radicals, after they have reacted further to form diamagnetic species.

This chapter is a joint effort that aims to provide an illustrative view of the current understanding of the relationship between free radicals and disease. This is a vast subject and while EPR has an important role in its study‚ the role of EPR is far from dominant. Therefore‚ instead of trying to provide a comprehensive review of the role of free radicals in medicine‚ we have chosen to provide an overview through some general remarks‚ followed by more detailed considerations of several illustrative examples in which EPR has been employed in attempts to understand the basis of specific pathophysiologies in which free radicals have a postulated role. The first author has drafted the overview section and the primary author(s) for each specific section are indicated in those sections. The terms EPR and ESR are used in accordance with the preference of the authors of each section.

The theoretical basis for expecting free radicals to be related to disease has evolved over the years. Initially the hypothesized links between free radicals and disease were based on loosely reasoned considerations that because free radicals are so reactive‚ and cells are so well organized‚ that the occurrence of free radicals in vivo would likely lead to disease. This type of reasoning became somewhat more focused on the role of free radicals in cancer (Swartz‚ 1979) . The current status of concepts of the role of free radicals in cancer is illustrated in the section drafted by Gutierrez.

The next phase of theories potentially linking free radicals and disease was based on the specific potential of free radicals to initiate strong oxidizing reactions. The fundamental rationale comes from the fact that living organisms include many highly oxidizable components‚ yet usually require the presence of oxygen in order to carry out basic metabolic functions. Interest in these aspects became greatly increased with the discovery of the natural occurrence of large amounts of superoxide dismutases in both the blood and cells. Subsequently a role of free radicals in almost every conceivable disease has been proposed on various theoretical grounds related to the potential for uncontrolled oxidations. Such theories have general support by observations that the extent of many pathologies can be modified by changing the availability of superoxide (especially by modifying the activity of superoxide dismutases) or the strongly oxidizing functions of macrophages. But the observations did not provide rigorous proof of causality‚ and the chemical reactions through which the superoxide caused the observed effects has remained obscure.

Until very recently‚ it has been assumed that the damage caused by free radicals occurred because of direct reactions with key molecules‚ damaging sufficient numbers of the key molecules to alter function. This has been puzzling when looked at critically‚ because when considering the specific free radicals that are expected to be involved‚ it has been difficult to construct plausible reaction schemes that would account for the observed effects. The hydroxyl radical and closely related species (e.g. the perferyl radical)‚ are so reactive that they must be generated on or immediately adjacent to the target molecules‚ because their diffusion distance is usually the distance to the next molecule. Therefore it would take very special circumstances for such radicals to be important in a disease. On the other hand‚ the superoxide radical is so unreactive that it is difficult to understand how it can lead to the very profound effects that clearly are associated with excess or inappropriate production of superoxide.

Because of recent advances in molecular biology‚ however‚ it now seems reasonable to expect that within the next few years we will have‚ finally‚ an adequate theoretical basis for understanding the roles of free radicals in biology. Then it will be much more feasible to develop methods to control or ameliorate the undesirable processes that they can cause.

The keys to the potential of much more effective progress have been the discoveries that oxidizing species and the amount of oxygen can have profound effects on gene activation and cell signaling. This has‚ finally‚ provided a potentially satisfactory mechanism for understanding the association of free radicals (and other oxidizing species) with pathophysiology. It has become clear that there are a large number of activators and modulators of genes and cytokines that respond to the presence of oxidizing species. Currently the understanding of the role of free radicals and other oxidizing species in cell signaling is still at an early stage‚ but rapid progress is likely as the cascades of effectors that are activated or inactivated by the amount of oxidizing species and/or oxygen are delineated. It seems likely that in many cases the origin of the free radicals will be from inappropriate responses of the naturally occurring defense systems‚ mediated through cell signaling. The old paradigm of deleterious actions of free radicals occurring through direct damage from the free radicals is likely to be applicable only in exceptional circumstances (e.g. ionizing radiation). Thus the experimental approaches in which roles of free radicals are likely to be important aspects for understanding probably will be in the arena of molecular biology‚ and the role of EPR in these studies may be critical. This is perhaps even more exciting than the vague postulates of free radicals being "bad" and leading to disease‚ because the responses of cells to the presence of oxidizing species are probably very fundamental properties of cellular systems. These mechanisms have enabled life to evolve in an environment with an abundant potential for deleterious oxidations‚ due to an atmosphere that is 20% oxygen. With this understanding‚ the value of EPR and related techniques to follow free radical reactions is likely to increase‚ as the techniques are applied in experiments that are more appropriately framed.

It is not surprising then‚ that recently theories suggesting links between free radicals and pathophysiological‚ physiological‚ and therapeutic processes have become more specific‚ linked to specific intermediates and/or specific cell signaling pathways. These are areas where it seems more likely that important and useful links will be found. EPR is likely to have an important role in elucidating what already has been shown to be a very complex set of processes.

Initially investigators sought evidence for the role of free radicals in disease by direct observations with EPR‚ reasoning that there should be increased amounts of free radicals in the related diseases. In general increases were not found‚ although in a few cases in studies of cancer some unexpected EPR signals were seen (Swartz‚ 1986). As noted in the review‚ more thorough consideration of this approach leads to the conclusion that even if free radicals are involved‚ there are seldom specific reasons for there to be increased amounts of free radicals that can be detected by EPR. The role of free radicals could be critical but transient. And even if there was a continuing role of free radicals‚ the more reactive that they are‚ the less likely it would be that they could be detected directly by conventional EPR spectroscopy. Hence it is not surprising that there has been little concrete evidence for a general link between the amount of free radicals and pathological or physiological processes.

Subsequently EPR has been used more specifically to detect free radical intermediates associated with a wide range of physiological‚ pathophysiological‚ and therapeutic processes that are potentially related to medicine. These studies have ranged from serially observing tissues in which tumors are expected to development in models of carcinogenesis‚ to looking for free radical intermediates in drugs. The more specific the hypothesis and the rationale for anticipating the occurrence of free radicals‚ the more likely it is that such approaches will be productive. In virtually all situations‚ it is unlikely that measurements of free radicals will be sufficient to test a hypothesis or reach a firm conclusion pertinent to a disease or its treatment. Appropriate additional studies‚ including tests of factors that may affect the amount or type of free radicals usually will be needed. The later sections of this chapter provide some illustrative examples of studies in which the role of free radicals in disease have been studied under appropriately rigorous conditions.

A wide and effective range of approaches have been used for studies in animal models relevant to clinical medicine. The available approaches are quite wide because unlike the situation in human subjects‚ there is much less concern about the long term effects of materials that are administered to carry out the studies and/or the invasiveness of the approach. Recently‚ as described in much more detail in ch. 9 on in vivo studies it has become possible to carry out studies in intact animals as well as in model systems and cells.

Sometimes it is possible to have direct detection of free radicals‚ if they occur in high enough concentrations and have sufficient stability. This approach is especially applicable when substantial amounts of a substance are administered‚ such as with therapeutic drugs or toxins. The occurrence of free radical intermediates of drugs is among the most likely situations where direct observations with EPR are likely to be productive. Sometimes it may be possible to have sufficient amounts of radicals to observe them directly in aqueous systems under physiological conditions‚ including normal temperatures for the organism (Fujii et al.‚ 1994; Mader et al.‚ 1995) . Often this will not provide sufficient sensitivity and rapid freezing will be required. While the use of frozen samples often results in the loss of resolvable structure in the EPR spectrum‚ the ability to have a much larger sample (because of the much lower dielectric loss in ice‚ the sample volume can be much larger) and the enhanced sensitivity of lower temperatures usually result in very significant increases in sensitivity versus aqueous samples.

For most studies‚ however‚ it is likely that there will be a need to employ spin trapping or other techniques to observe the free radicals. Several different experimental approaches using spin traps have been successfully employed with cells and experimental animals including: a) use of model systems (in vitro) . This is the most widely used approach.

The concentration of spin traps can be made as high as needed when live organisms are not used. When the studies are carried out in cell systems careful attention has to be paid to effects of the spin traps on the cells (toxicity) and effects of the cells on the spin adducts (stability of the adduct) (Khan et al.‚ 2003) . b) ex vivo approaches in which the trapping occurs in vivo and samples are obtained for study in vitro. This approach has been used productively, especially by Mason and colleagues (ch. 5) . It involves the administration of the spin trap to the animal and then obtaining samples through biopsies or removal of biological fluids. It can provide excellent evidence for the occurrence of free radical reactions in vivo, but considerable care is needed to avoid artifacts or misleading results. The potential problems include loss of spin adducts during the extraction steps and generation of spin adducts during the processing of the samples. c) direct spin trapping in vivo with measurements made directly in the organism under physiological conditions. In principle this is a very desirable approach, providing direct evidence without intervening processing steps and, also, the potential to provide kinetic data under physiological conditions. This approach, however, has lower sensitivity than ex vivo methods, because the latter can study the samples at X-band or even higher frequencies‚ while direct in vivo studies require the use of L-band or even lower frequencies‚ with the consequent power sensitivity of such frequencies. Both ex vivo and direct in vivo approaches share the problem of potential instability of the spin adducts‚ which often are converted to non-paramagnetic products in the presence of functioning cells (Khan and Swartz‚ 2002; Timmins et al.‚ 1999) . spin trapping using combined NMR and EPR. This is a new approach whose utility has not been fully evaluated. In principle it provides a means to use the widespread availability of in vivo NMR‚ and that is very attractive. The section by Berliner in this chapter provides an excellent summary of this approach. indirect assays based on reactions of free radicals with nitroxides or other paramagnetic labels. This has been especially advocated by Utsumi's group‚ using the rate of disappearance of nitroxides. While in principle this could be a very productive approach‚ there are some potential fundamental limitations because of the non-specificity of the observed parameter.

d) e)

In the near future it may be possible to develop techniques for studies in human subjects. The potential limiting factors for such studies include the technical problems of carrying out EPR measurements in human subjects and‚ for techniques that involve the administration of spin traps or other substances‚ the complex and difficult process for obtaining permission to administer substances to human subjects (Swartz‚ 2003) . These also are discussed in some detail in ch. 9. Here we will briefly summarize those aspects that are especially pertinent to measurements of free radicals in human subjects.

The technical problems for using EPR in human subjects revolve around accommodating the human body in the system and obtaining sufficient sensitivity. The geometric constraints can readily be overcome and at least one specifically designed whole body clinical EPR spectrometer already is in operation. This is at Dartmouth where a conventional but large-sized permanent magnet has been specifically built by Sumitomo Special Metals for the facility. It provides a 400 gauss field with a gap that can accommodate human subjects on a cart‚ or sitting or standing within the gap of the magnet.

For studies in which the direct observable is a free radical form of a drug‚ etc.‚ the only potential limitations are the sensitivity that can be achieved and the pertinence of the radical to the processes under investigation. Unfortunately‚ there appears to be a very limited number of drugs or toxins that generate sufficient amounts of free radicals to be observed directly.

All other approaches‚ e.g. spin traps‚ interactions with nitroxides‚ require the administration of a compound. This adds the requirement that the administered compound has been demonstrated to be safe for use in humans. This is potentially a very limiting aspect‚ because of the time and expense involved in obtaining the data for achieving such clearance.

There are a few drugs that may be of use that already have been cleared for use in human subjects. In particular‚ one of the usual EPR responsive trapping agents for nitric oxide‚ the dithiocarbamates‚ have been approved for use in humans as chelators of potentially toxic metals and as the alcohol avoidance agent ("antabuse").

There are some possibilities for the approval of some spin trapping agents and nitroxides as the result of initiatives to use them as therapeutic agents. The nitrone spin traps are being evaluated for use in humans because they appear to have potential therapeutic benefits‚ probably through their effects on the induction of nitric oxide synthesizing enzymes. The nitroxides have been put forth as potential therapeutic agents for the dismutation of superoxide and/or as protective gents against damage by ionizing radiation. If these initiatives result in the approval of nitrones or nitroxides for use in humans‚ then it would be much more feasible to obtaining permission to use them as spin trapping agents in humans.

PULMONARY FREE RADICAL DAMAGE (R. MASON)

The herbicide paraquat serves as a model for investigating free radicalmediated pulmonary toxicity because it has no known metabolism other than the free radical metabolism. In microsomal systems‚ the enzymatic reduction of paraquat to its cation radical is catalyzed by the flavoenzyme NADPHcytochrome P-450 reductase. The paraquat radical is stable in the absence of oxygen. In the presence of oxygen‚ paraquat is re-formed‚ and superoxide is generated in a catalytic fashion with no net change occurring to the paraquat molecule. This process has been termed futile metabolism (Mason 1979‚ 1982 . The mechanism of paraquat poisoning in man is a superoxidemediated toxicity that is completely analogous to the herbicide mode of action. The lung is the site of injury by paraquat because it accumulates there (Rose and Smith‚ 1977) . The energy-dependent uptake of paraquat and the subsequent free radical formation are cell-specific. Paraquat free radical formation occurs with clara cells and alveolar type 2 cells‚ but not with alveolar macrophages (Horton et al.‚ 1986) . Diquat‚ morfamquat‚ and other bipyridylium compounds do not affect the lung as seriously‚ but these compounds do cause liver damage and are reduced by rat hepatocytes to their respective radical cations (DeGray et al.‚ 1991).

In early investigations of the mechanism of rat hepatic mitochondria! and microsomal nitroreductase (Mason and Holtzman‚ 1975 a) ‚ ESR and kinetic evidence demonstrated that the first step in these nitroreductase reactions is the transfer of a single electron to nitro compounds to give the corresponding nitro anion free radical. For instance‚ in the case of nitrofurantoin‚ the interaction of the free electron with the nitrogens and protons gives a complex hyperfine pattern that has been analyzed to demonstrate that the free radical is simply nitrofurantoin plus an extra electron (Rao et al.‚ 1987‚ 1988a .

Nitrofurantoin increased the NADPH-supported oxygen consumption by pulmonary microsomes sevenfold over the basal rate‚ and this stimulation was partially reversed by superoxide dismutase. The presence of superoxide anion radical strongly suggested reductase under aerobic conditions (Mason and Holtzman‚ 1975b) . As expected‚ the disproportionation of hydrogen peroxide by catalase also decreased the nitrofurantoin-stimulated oxygen uptake.

The effect of superoxide dismutase and catalase on the nitrofurantoinstimulated oxygen consumption by microsomes is consistent with the formation of nitroaromatic anion radicals under aerobic conditions and with the rapid air oxidation of these radical intermediates‚ which results in the catalytic generation of superoxide and the well-known oxygen inhibition of nitroreductases. The nitrofurantoin-catalyzed reduction of oxygen to superoxide and the hydrogen peroxide that forms from this superoxide may be responsible for some of the toxic manifestations that occur during nitrofurantoin therapy (Mason and Holtzman‚ 1975b) . For instance‚ the occasional cases of pulmonary edema and fibrosis caused by nitrofurantoin therapy are similar to the effects of paraquat poisoning. Subsequent work with animal models supported this proposal (Peterson et al.‚ 1982; Boyd et al.‚ 1979) . Boyd and coworkers (1979) have shown that the acute toxicity of nitrofurantoin is markedly increased by vitamin E deficiency‚ an oxygenenriched atmosphere‚ or a diet high in highly polyunsaturated fats. Further studies by Peterson and colleagues (1982) showed that decreasing the activity of selenium-dependent glutathinone peroxidase in 8-day-old chicks enhanced the acute toxicity of nitrofurantoin.

Both cigarette smoke and the smoke from burning buildings form free radicals even after combustion stops. Both the in vitro effects of this smoke and the use of ESR in these studies have been extensively reviewed (Pryor 1992) . Smoke inhalation by rabbits formed unidentified PBN radical adducts in plasma (Yamaguchi et al.‚ 1992; Murphy et al.‚ 1991) . Cotton smoke formed radical adduct concentrations similar to those caused by 2.5 atm of oxygen (Yamaguchi et al.‚ 1992) . Formation of a burst of PBN radical adducts by a brief exposure to cigarette smoke required pretreatment by bacterial endotoxin (Murphy et al.‚ 1991) .

The spin trapping technique detected free radicals produced in vivo by ozone exposure (Kennedy et al.‚ 1992) . When rats were exposed for 2 hr to either 0‚ 0.5‚ 1.0‚ 1.5‚ or 2.0 ppm ozone with 8% to increase their respiratory rate‚ a six-line 4-POBN/radical spin adduct signal was detected by ESR in lipid extracts from lungs of rats treated with 4-POBN. Only a weak signal was observed from rats exposed to 0 ppm ozone (air with only). A correlation was observed between the radical adduct concentration and the lung weight/body weight ratio‚ an index of lung damage. These results demonstrate that ozone induces the production of free radicals in rat lungs during inhalation exposure and that radical production may be involved in the induction of pulmonary toxicity by ozone.

It has been postulated that the in vivo toxicity of asbestos results from its catalysis of free radical generation. We examined in vivo radical production using ESR and the spin trap 4-POBN; 180 day-old rats were intratracheally instilled with either crocidolite asbestos or saline. Twenty-four hours later‚ histologic examination revealed a neutrophilic inflammatory response. ESR spectroscopy of the chloroform extract from lungs exposed to asbestos gave a spectrum consistent with a carbon-centered radical adduct‚ while those spectra from lungs instilled with saline revealed a much weaker signal. This same radical formation persisted and‚ even one month after instillation‚ could be detected in the lungs of rats exposed to asbestos.

Exposure to air pollution particles can be associated with increased human morbidity and mortality. Lung exposure to oil fly ash (an emission source air pollution particle) causes in vivo free radical production as evidenced by ESR analysis of chloroform extract from lungs of animals exposed to oil fly ash‚ which gave a spectrum consistent with a carboncentered radical adduct (Kadiiska et al.‚ 1997) . This signal was also reproduced by instilling animals with the soluble fraction of the oil fly ash‚ which contains soluble metal compounds. The same signal was observed after instillation of either a mixture of vanadium‚ nickel‚ and iron sulfates or alone‚ metals which are prevalent in oil fly ash. Therefore‚ this generation of free radicals appears to be associated with the soluble metals in the oil fly ash.

Intratracheal instillation of lipopolysaccharides (LPS) activates alveolar macrophages and infiltration of neutrophils‚ causing lung injury/acute respiratory distress syndrome. Free radicals are a special focus as the final causative molecules in the pathogenesis of lung injury caused by LPS. In vivo free radical production by rats was detected after intratracheal instillation of LPS. ESR spectroscopy of lipid extract from lungs exposed to LPS for 6 h gave a spectrum consistent with that of a POBN/carbon-centered radical adduct tentatively assigned as a product of lipid peroxidation. To further investigate the mechanism of LPS-initiated free radical generation‚ rats were pretreated with the phagocytic toxicant which significantly decreased the production of radical adducts with a corresponding decrease in neutrophil infiltration. NADPH oxidase knockout mice completely blocked phagocyte-mediated‚ ESR-detectable radical production in this model of acute lung injury‚ demonstrating that superoxide formation by NADPH oxidase is the root cause of this free radical formation.

There has been a long association between sickle-cell disease and the formation of oxidants from the partial reduction of molecular oxygen. In general this association has focused on either the deposition of redox active iron as a mediator of biomolecule oxidation‚ or on accelerated autoxidation of HbS as opposed to HbA‚ leading to enhanced superoxide formation. In both cases increased oxidative stress in sickle cell disease results in the oxidation of biological macromolecules which contributes to the pathogenesis of the disease (Hebbel et al.‚ 1982; Klings et al.‚ 2001; Natta et al.‚ 1992; Repka and Hebbel‚ 1991; Schacter‚ 1986; Sheng et al.‚ 1998) of free radicals and oxidants in the control of vascular function (as opposed to oxidative damage).

Endothelial dysfunction‚ which is most often indicated by loss of endothelial-dependent relaxation‚ can be regarded as a disease of altered free radical balance. For example the scavenging of nitric oxide by superoxide has long been thought to be contributing factor to hypertension and it is becoming apparent that this same mechanism may be responsible for altered endothelial signaling in atherosclerosis‚ diabetes‚ and other cardiovascular pathologies‚ and may also be a control mechanism of normal cardiovascular responses such as the modulating effects of shear stress on vessel relaxation. Recently two nitric oxide scavengers have been identified in the lumen of patients with sickle cell disease.

The first of these‚ xanthine oxidase‚ has long been understood to be a physiological source of superoxide‚ however‚ the recently discovered elevation of vessel wall xanthine oxdase may be an important component of endothelial dysfunction in sickle cell disease. Aslan et al. (2001) have demonstrated that liver injury in both sickle-cell patients and a mouse sickle cell model can result in the release of xanthine oxidase into the blood stream. Accumulation and/or uptake of this enzyme by the vascular endothelium can lead to enhanced superoxide production and impairment of endotheliumdependent relaxation.

Another molecule that has only recently been implicated as a nitric oxide scavenger in sickle cell disease is plasma hemoglobin. A major paradox of nitric oxide function has been the presence of high (10 mM) concentrations of hemoglobin in the vascular lumen. Calculations have clearly shown that this level of hemoglobin should preclude nitric oxide radical from having any appreciable steady-state concentration in the vessel wall. This paradox has been recently addressed by the discovery that compartmentalized hemoglobin reacts with nitric oxide at significantly slower rates (up to 1000 times) than cell-free hemoglobin. The effects of increased levels of cell-free hemoglobin on vascular function have only recently been realized. As recently demonstrated by Reiter et al. (2002) ‚ the levels of plasma cell free hemoglobin are substantially elevated in patients with sickle cell disease‚ averaging and as high as The hemoglobin appears to be in the ferrous form as the plasma from sickle cell patients exhibits a greater ability to scavenged nitric oxide. Measurements of blood flow in the forearm indicate that plasma hemoglobin can severely limit nitric oxide-dependent vasodilation. An important corollary of these studies is that pharmacologically administered nitric oxide (for example by inhalation) can act to 'scavenge the scavenger' by oxidizing plasma hemoglobin to its met or ferric derivative. Ferric hemoglobin then has limited ability to scavenge peripheral nitric oxide‚ thus potentially restoring NO bioavailablility. The level of methemoglobin in plasma (as demonstrated by electron paramagnetic resonance spectroscopy) is increased when sickle-cell patients inhale nitric oxide‚ together with the level of nitrosylhemoglobin (Reiter et al.‚ 2002) . Cell-free hemoglobin circulating through the pulmonary circulation is a much more amenable target for nitric oxide therapy than guanylyl cyclase and it may be the case that the effects of nitric oxide inhalation in other clinical conditions are at least in part mediated through plasma hemoglobin oxidation. It is interesting to speculate that the ability of hydroxyurea‚ recently identified as an NO congener‚ to relieve sickle cell disease may also be at least in part related to its ability to oxidize hemoglobin in the extracellular compartment (Glover et al.‚ 1999; Huang et al.‚ 2002; Jiang et al.‚ 1997) .

Motor neuron disease or ALS (also known as Lou Gehrig's disease) is a fatal illness that progressively causes the degeneration of the nerve cells that control the muscles‚ paralyzing the body but sparing the mind (Price et al.‚ 1994) . Approximately 10% of ALS cases are familial with the rest being sporadic. The genetic defect in familial ALS (FALS) has now been linked to Sod 1‚ the gene encoding the cytosolic Cu‚ Zn‚ superoxide dismutase (SOD1) (Brown‚ 1995‚ 1998 . At present‚ approximately 70 or so missense FALS mutations in Sod 1 at 26 different amino acid positions have been identified. The mechanisms by which FALS-linked Sod 1 mutants cause selective degeneration of motor neurons of the spinal cord‚ brain stem‚ and cerebral cortex remain unclear (Brown‚ 1995‚ 1998 . Of the various mechanisms proposed‚ the hypothesis suggesting that the FALS-linked mutants acquired a cytotoxic "gain-in-function" has found increased acceptance (Brown‚ 1998) . The "gain-in-function" of Sod 1 mutants had been postulated to arise from an increased peroxidase or hydroxyl radical activity. The EPR technique has played a key role in identifying the structure of the oxidant formed from the peroxidase activity of SOD 1 (Singh et al.‚ 1998) .

Nearly 30 years ago‚ Hodgson and Fridovich demonstrated that the copper-bound hydroxyl radical a putative oxidant generated in the reaction between SOD1 and could oxidize several anionic ligands (e. g.‚ formate‚ azide‚ and nitrite anion) (Hodgson and Fridovich‚ 1975) . In addition to these anions‚ several other molecules (which are too bulky to find access to the active site of Cu‚ Zn and SOD) were also oxidized. These experiments were performed in a bicarbonate buffer.

The x-ray crystal structure of SOD1 indicates that access to the active site is via a narrow channel that restricts the entry of large molecules. However‚ a relatively small anion like could reach the active site of SOD1 and undergo oxidation to the carbonate anion radical by is a diffusible oxidant that could leave the active site and cause oxidation of various substrates in free solution. Bicarbonate thus effectively exports oxidation from the sterically hindered active site to large molecules in bulk solution (Liochev and Fridovich‚ 1999) . This model which gives a new perspective on the peroxidative mechanism of SOD1‚ is pivotal for explaining the published discrepancies in spin trapping reports using SOD1 and FALS SOD1 mutants (Goss et al.‚ 1999; Zhang et al.‚ 2002) .

Evidence for increased peroxidase activity from FALS SOD1 mutants (e. g.‚ A4V with alanine substituted by valine at amino acid 4‚ G93A with glycine replaced by alanine at amino acid 93) was first obtained by monitoring the oxidation of nitrone spin trap 5‚ 5'-dimethyl-1-pyrroline Noxide (DMPO) to its hydroxylated adduct (DMPO-OH) (Wideau-Pazos et al.‚ 1996) . The EPR spectrum of DMPO-OH is characterized by an equivalent hyperfine interaction (=15 G) from the nitroxide nitrogen and the atom. Investigators attributed the formation of DMPO-OH to trapping of hydroxyl radical (see Zhang et al.‚ 2000 for references). Significant differences in DMPO-OH signal intensity were observed between the wild type SOD1 and FALS mutant SODs. However‚ the fact that bicarbonate was needed for DMPO-OH formation in this system has been previously ignored.

We reinvestigated the mechanism of formation of DMPO-OH using oxygen-17 labeled water and hydrogen peroxide. Results from these experiments unambiguously demonstrated that nearly all of the oxygen in DMPO-OH originated from water. We proposed that either a nucleophilic addition of to the DMPO-carbonate radical intermediate or an addition of to the DMPO cation radical intermediate. Independent evidence for the intermediacy of was obtained from photolysis studies using the pentammine carbonate complex of Co (III). The UV photolysis of this cobalt complex has been shown to generate the authentic radical. This system yielded the same type of radical adducts in the presence of substrates (e. g., azide, ethanol, and formate) as detected in the system (Zhang et al., 2000 (Zhang et al., , 2002 . Bicarbonate-dependent peroxidase activity of SOD1 was found to be responsible for hydroxylation and oxidation of azulenyl nitrone to an aldehyde. Aldehyde formation was attributed to trapping of hydroxyl radicals by the azulenyl nitrone (Gurney et al., 1998) . Recently, we showed that in the absence of bicarbonate, oxidation of azulenyl nitrone to azulenyl aldehyde was negligible in phosphate buffers containing SOD1, and the chelator DTPA (Zhang et al., 2002) . Photolysis of solutions containing the cobalt complex that generates the carbonate radical anion and azulenyl nitrone yielded azulenyl aldehyde. Based on these results, we proposed that is responsible for mediated oxidation of azulenyl nitrone. Bicarbonate-dependent SOD1 peroxidase activity is, thus, inhibitable by nitrones. Bicarbonate-dependent SOD1 peroxidase activity was shown to be higher in FALS mutants (Brown, 1995 (Brown, , 1998 .

The first in vivo spin trapping evidence for increased free radical formation was provided using the SOD1-G93A transgenic mouse model for FALS (Gurney et al.‚ 1998) . The investigators found that when azulenyl nitrone was administered to the nontransgenic‚ wild-type transgenic‚ and mutant transgenic mice of different ages (30‚ 60‚ and 90 days)‚ increased levels of azulenyl aldehyde were detected in spinal cord extracts of mutant mice but not in transgenic wild-type and nontransgenic mice. Concomitantly‚ azulenyl nitrone treatment prolonged the survival of FALS overexpressing mice.

More recently‚ it was shown that treatment of mutant G93A-SOD1 transgenic mice with nitrone trap DMPO significantly delayed paralysis and prolonged survival (Li et al.‚ 2002) . However‚ DMPO-derived products were not analyzed. In conclusion‚ in vitro and in vivo experiments suggest that nitrone spin traps can potentially mitigate oxidative stress in FALS mutant overexpressing cells and mice and protect against progressive motor neuron death.

Melanins are virtually the only naturally occurring stable free radicals in mammals. EPR spectroscopy can be used as a unique non-destructive tool for studying melanin in in vitro in cultivated cells (Pilas and Sarna‚ 1985; Cieszka et al.‚ 1995; Hill et al.‚ 1997) ‚ in isolated tissues and organs ex vivo (Enochs et al.‚ 1993a; Slominski et al.‚ 1994; Plonka et al.‚ 2002) ‚ and even under in vivo conditions (Lukiewicz and Sarna‚ 1972; Katsuda et al.‚ 1990) . The putative function of the pigment is protection against light-induced damage‚ perhaps mediated by free-radical damage (Woods et al.‚ 1999) .

The interactions of melanin probably involve both radical and nonradical aspects of the quinone-hydroquinone equilibria that underlie many of its properties (Sarna and Swartz‚ 1993) . Because of their redox activity and ability to bind charged materials‚ including metal ions and drugs‚ melanins potentially can affect many physiological‚ pathophysiological‚ and therapeutic processes (Enochs et al.‚ 1994; Larsson‚ 1993; Mars and Larsson‚ 1999) . In addition‚ due to the high reactivity of some of the intermediates‚ melanin synthesis has also been linked to cytotoxicity (Halaban and Lerner‚ 1977; Pawelek and Lerner‚ 1978) .

Because of these complex and diverse properties‚ the study of melanins with EPR has been a large and productive field that has pertinence to the subject of this chapter: free radicals and medicine. As an example of such studies‚ we consider here two aspects in which the ESR Center at the Medical College of Wisconsin has been especially involved‚ ocular melanins and neuromelanins.

Specialized cells that synthesize melanin in humans are found not only in the skin epidermis and in the hair follicles‚ they are also present in the eye‚ ear and in restricted regions of the brain (Boissy‚ 1998). Although populations of these extracutaneous melanin-synthesizing cells have some morphological and functional similarities to the various cutaneous melanocytes‚ it is expected that significant differences between extracutaneous and cutaneous melanocytes should exist (Boissy‚ 1998). Melanin synthesis usually occurs in specific organelles‚ the melanosomes‚ and is controlled by specialized enzymes (Orlow‚ 1998;  Pawelek and Chakraborty‚ 1998)‚ but the formation of neuromelanin (NM) in dopaminergic and noradrenergic neurons of the substantia nigra (SN) and locus coeruleus (LC)‚ appears to be enzyme-independent and the neuromelanin granules show significant similarities to inactivated lysosomes (Barden and Brizzee‚ 1987) .

In primates‚ two different cell types are involved in ocular melanogenesis -melanocytes of the uveal tract and neuroepithelial cells of the retinal pigment epithelium (RPE). All true melanocytes‚ including ocular melanocytes‚ originate from the neural crest; in contrast‚ the RPE cells are derived from neuroepithelial cells of the developing forebrain of the embryo (Noden‚ 1991; Mann‚ 1964) . Both types of the melanin-synthesizing cells of the eye express melanocyte-specific proteins (Boissy‚ 1998).

Allthough biological functions of ocular melanin have not been unambiguously determined‚ a growing body of experimental evidence and epidemiological data suggest that both the iridial (uveal) and RPE melanin may have important photoprotective and antioxidant properties (Sarna‚ 1992; Sarna and 1994) . Clearly‚ ocular pigmentation‚ particularly in the uveal tract‚ protects the retina from overexposure by absorbing and scattering the impinging light. Melanin in the choroid and RPE contributes to visual acuity by preventing light reflection from the fundus. It has long been recognized that the severity of the visual abnormalities in albinism correlates with the degree of hypopigmentation (Kinnear et al.‚ 1985) . However‚ even with corrective tinted lenses that help photophobia and‚ in a minor way‚ vision by increasing contrast‚ the results in albinos are rather disappointing (Taylor‚ 1978) . It has been postulated that the albino visual system may represent a case of arrested development (Wilson et al.‚ 1988) . Thus‚ melanin synthesis in the RPE appears to be of crucial importance for normal development of the visual system of an organism‚ and the presence of RPE is not only responsible for the maintenance of the neural retina but also for its successful embronic development and organization (Boissy et al.‚ 1993; Raymond and Jackson‚ 1995) .

Age-related macular degeneration (AMD) is the major cause of late onset blindness in developed countries (Evans and Wormald‚ 1996; Klein et al.‚ 1992) . There is currently no established etiology that could serve as a basis for preventive medicine and no cure for this disease. Light can damage photoreceptor cells in the mammalian retina (Organisciak and Sarna‚ 2001; Rapp‚ 1995; Ham et al.‚ 1980) and population-based studies indicate an association between advanced AMD and a patients' exposure to blue or visible light over the preceding 20 years (Cruickshanks et al.‚ 1993; McCarty and Taylor‚ 1999; Taylor et al.‚ 1992) . The primary lesion in AMD may occur in the RPE (Zarbin‚ 1998) and result from oxidative damage (Beatty et al.‚ 2000; Cai et al.‚ 2000) .

Due to the close structural and functional association between the RPE and photoreceptors‚ it is generally agreed that any oxidative damage to the RPE‚ which causes RPE dysfunction‚ could contribute to the photoreceptor degeneration that characterizes AMD. Thus‚ the antioxidant status of the RPE may be a key factor in determining the tissue's health‚ and the health of the photoreceptors that the RPE supports. An RPE antioxidant that may be particularly relevant when photic stress is implicated in tissue damage is the light-absorbing pigment melanin (Sarna‚ 1992). It has been postulated that RPE melanin can protect the RPE from oxidative stress by sequestering redox active metal ions‚ quenching electronically excited states of certain photosensitizing dye molecules and by scavenging reactive free radicals (Sarna et al.‚ 1998; Rozanowska et al.‚ 1999) . Significantly‚ melanin content of the human eye seems to be inversely correlated with the incidence of AMD (Weiter et al.‚ 1985; Young‚ 1988 )‚ and the risk of suffering from AMD is about 40 times higher in whites than in blacks (Pauleikhoff and Holz‚ 1996) ‚ even though the inter-racial differences in the amount of ocular melanin mainly concern the uveal pigmentation. However‚ it is important to realize that antioxidant abilities of melanin may change with age. Indeed‚ a recent EPR study has shown that the amount of RPE melanin decreases by a factor of 2.5 between the first and ninth decade of life (Sarna et al.‚ 2002) . In an independent study‚ the melanin content of RPE melanosomes from human donors of different age was analyzed by EPR after drying the pigment granules (Bilinska et al.‚ 2002) . Surprisingly‚ the content of melanin‚ when normalized to a single pigment granule‚ was found independent of age. On the other hand‚ RPE melanosomes from younger individuals could contain significantly higher percentage of strongly bound water‚ that those from older individuals (Bilinska et al.‚ 2002) . RPE melanosomes from older human donors exhibit increased photoreactivity: they induce faster oxygen photouptake and accumulation of superoxide anion spin adducts (Rozanowska et al.‚ 2002) . Thus with age‚ RPE melanin may contribute to oxidative stress in the outer retina both as a result of a decreased antioxidant efficiency and an increased aerobic photoreativity. Interestingly‚ W-band EPR spectroscopy may provide a sensitive measure for differentiating RPE melanin from human donors of different age and monitoring possible chemical changes of the melanin that are likely to be aggravated with aging (Rozanowska et al.‚ 1993) . In this preliminary study a striking pattern in the changes of magnetic parameters of melanin radicals with age was observed. Although the molecular nature of these changes is not yet understood‚ they may result from life-long oxidative modifications of the RPE melanin.

Neuromelanin (NM) is an insoluble brown-black or greyish in appearance pigment that accumulates with age in the midbrain of primates‚ most notably of humans (Van Woert et al.‚ 1966) . Based on standard histochemical tests and various physicochemical analysis‚ including EPR spectroscopy‚ NM has been classified as a true melanin (Barden and Brizzee‚ 1987; Enochs et al.‚ 1993b) . Characteristic degradation products of NM indicate that this melanin has chemical properties similar to both eumelanins and pheomelanins (Odh et al.‚ 1994) . It appears that the synthesis of NM occurs via oxidative polymerization of dopamine and its sulfur containing derivatives. As a synthetic model of NM‚ a polymer obtained by autooxidation of dopamine and cysteine is often used (Shima et al.‚ 1997) . Biological functions of NM remain unknown. Unlike cutaneous and ocular melanins‚ neuromelanin is never exposed to environmental light. Therefore‚ no photoprotective role of NM is likely.

It has been postulated that under normal physiological conditions NM may have a cytoprotective function by the sequestration of redox-active metal ions (Swartz et al.‚ 1992; Zareba et al.‚ 1995; Korytowski et al.‚ 1995) . On the other hand‚ the presence of NM may also be responsible for the vulnerability of pigmented neurons observed in Parkinson's disease (PD). This neurological disorder is characterized clinically by akinesia‚ rigidity‚ and tremor (Stern‚ 1990) . Histological analysis of Parkinsonian brains shows extensive degeneration of the pigmented dopaminergic neurons of the substantia nigra. In PD‚ the neuromelanin containing cells of the SN appear to be more vulnerable to degeneration than the non-pigmented neurons. However‚ there seems to be no strict correlation between the vulnerability of the neurons and their melanin content (Kastner et al.‚ 1992) .

The formation of NM in the human brain‚ probably is a by-product of oxidative metabolism of dopamine‚ known to occur at high rate in catecholaminergic neurons of the SN and LC. While both autooxidative reactions and enzymatic oxidation of dopamine are accompanied by the generation of hydrogen peroxide‚ the former process can also lead to the formation of potentially cytotoxic semiquinones and quinones (Cohen‚ 1983; Graham 1984) . Therefore‚ the dopaminergic neurons of the SN and LC are intrinsically at risk of oxidative stress. Despite the presence of antioxidative mechanisms in catecholaminergic neurons for maintaining their neurotransmitters in a reduced state‚ the system is not perfect and some semiquinones and quinones may escape from reduction and randomly form products that cannot be catabolized and gradually accumulate in lysosomes‚ eventually becoming neuromelanin granules (Enochs et al.‚ 1994) . It can be argued that when formed‚ NM can exert its antioxidant action by binding of advantitious multivalent metal ions‚ such as iron and copper (Swartz et al.‚ 1992) . There is evidence that SN neurons in patients with PD exhibit an elevated level of oxidative stress (Jenner‚ 1991). Thus the ratio of reduced to oxidized glutathione is decreased in Parkinsonian brains‚ compared to normal brains (Riederer et al.‚ 1989; Sofic et al.‚ 1992) .

In addition‚ a decreased level of polyunsaturated fatty acids and increased levels of malondialdehyde‚ a product of lipid peroxidation‚ were found in PD brains (Dexter et al.‚ 1989) . Whether these changes are the cause of the disease or they result from an increased dopamine turnover in the remaining neurons of a PD patient‚ remains unclear.

One of the most important properties of NM that may determine its biological functions is the ability of NM to bind metal ions (Enochs et al.‚ 1993b) . High levels of metal ions‚ particularly iron; were found in the human SN (Zecca and Swartz‚ 1993; Gerlach et al.‚ 1995; Shima et al.‚ 1997) . Although ferritin is known to be responsible for accumulation of iron in many tissues‚ it is believed that in SN iron is bound to NM Zecca and Swartz‚ 1993; Gerlach et al.‚ 1995) . Indeed‚ recent measurements suggest that ferritin is not present in SN neurons (Moos‚ 2000)‚ which implies that neuromelanin may be a key buffering system for iron in pigmented neurons (Zecca et al.‚ 2002) .

The antioxidant efficiency of neuromelanin was studied in model systems. The study has clearly shown that the yield of hydroxyl radicals generated via iron ion-catalyzed free radical decomposition of hydrogen peroxide dramatically decreased in the presence of synthetic NM (Zareba et al.‚ 1995) . Both synthetic and natural NMs were able to inhibit peroxidation of lipids induced by iron/ascorbate or thermolabile azo-compounds . A hypothesis‚ about the role of NM in the multifactioral etiology of PD was proposed by Enochs et al. (1994) : oxidative degradation of NM that can occur with aging or as a result of an intense or chronic oxidative stress in the pigmented neurons of SN could‚ via positive feed-back mechanism‚ further increase the level of the oxidative stress. Oxidatively modified NM is expected to have a decreased binding affinity for iron and other metal ions. It would‚ therefore‚ not only bind fewer metal ions compared to "native" NM‚ but even raise the cytoplasmic level of potentially cytotoxic species by release of the accumulated ones.

The genetic basis of human cancer has been well documented. Widespread genomic instability is a hallmark of tumor cells (Jeong‚ 2003; Nowell‚ 1976 ). An average of about 1/3 of all tumor types appear to exhibit a genomic instability manifested by subtle nucleotide sequence alternations that activate proto-oncogenes or inactivate tumor suppressor genes. Chromosomal rearrangements are likely to be a result of aberrant recombination repair that occurs during normal cellular mitotic growth (Przybytkowski‚ 2003; Friedberg‚ 1985; Friedberg et al.‚ 1995; Kucherlapati and Smith‚ 1988) . A very large number of genes has been identified as tumor promoter genes or tumor suppressor genes (Cheng‚ 2003; Stanbridge‚ 1990; Weiberg‚ 1989) . As far back as 1993‚ genetic analysis of tumor cells has suggested that between 6 to 12 of these genes are altered during the development of a tumor (Renan‚ 1993). The number of altered genes far exceeds the number of mutations that would be predicted by the spontaneous mutation rate in human cells. Of the several theories to account for this discrepancy‚ the "mutator hypothesis" for tumorigenesis has gained much attention (Loeb‚ 1991‚ 1994 . In this hypothesis‚ an early spontaneous mutation in one of many genes that maintain the genetic information in chromosomes becomes altered. This leads to genetic instability‚ or a mutator phenotype‚ either by accumulation of replicative errors or by gross chromosome rearrangement. A higher-than-normal mutation rate then leads to the combination of mutations in genes that regulate growth‚ invasion‚ or metastasis. Post-replication mismatch repair systems enhance the fidelity of DNA replication by correction of replicative errors. It has recently been shown that mutations in mismatch repair genes are associated with cancers (e.g. Jeong‚ 2003; Worrillow‚ 2003) .

Oxidative stress caused by reactive oxygen species (ROS) can lead to genomic instability. Oxygen is metabolized inside the cell by a series of one electron reductions with the generation of reactive and potentially damaging ROS which include superoxide radical anion‚ and the hydroxyl radical (Halliwell and Gutteridge‚ 1989) . A pro-oxidant status in the cell can lead to the accumulation of ROS. Humans are exposed to substantial amounts of oxidative stress from a variety of sources. Halliwell has estimated levels of up to in the urine of individuals ingesting such foods as instant coffee (Long et al.‚ 1999a‚b) .

Oxygen and its metabolites are known to function as intracellular signals‚ playing a role in apoptosis (Butts et al‚ 2003; Liu et al.‚ 2003; Barr and Tomei‚ 1994; Sandstrom‚ 1994‚1995; Corcoran et al.‚ 1994; Davies‚ 1995; Forrest et al.‚ 1994; Greenspan and Aruma‚ 1994; Hockenbery‚ 1995; Sandstrom et al.‚ 1994) 

and metabolizing enzymes (Anderson et al.‚ 1994; Burdon‚ 1995; Crawford et al.‚ 1988; Curran and Morgan‚ 1995; Janknecht et al.‚ 1995; Khan and Wilson‚ 1995; Koong et al.‚ 1994 a‚b; Chen and Giaccia‚ 1994; Nose et al.‚ 1991; Piechaczyk et al.‚ 1994; Remade et al.‚ 1995; Schreck et al.‚ 1991; Yao et al.‚ 1994; Lambert et al.‚ 1994; Vile et al.‚ 1994; Firth et al.‚ 1994) . ROS are believed to play a causative role in the degenerative diseases of aging including cancer Peterszegi et al.‚ 2003; Cantuti-Castelvetri et al.‚ 2003) . Endogenously‚ activated neutrophils and macrophages generate large quantities of NO‚ as part of the inflammatory response (Chanock et al.‚ 1994; Densen et al.‚ 1995‚ Ginsburg and Kohen‚ 1995; Laskin and Pendino‚ 1995) ‚ and a variety of other normal cells can also be stimulated to produce low levels of superoxide and hydrogen peroxide (Burdon‚ 1995). Ames has estimated that one third of the cancers in developing countries can be attributed to chronic infections . When oxidative stress due to these reactive species from whatever source exceeds the capacity of the defense systems of the cells to intercept and neutralize them (i.e. prooxidant status)‚ toxicity (Mytar et al.‚ 1999) ‚ genomic instability and mutations in cells result. For example‚ tumor promoters stimulate the production of reactive oxygen species which may play a critical role in the progression to malignancy (Beckman et al.‚ 1994; Gopalakrishna et al.‚ 1994; Hu et al.‚ 1995; Marnett and Ji‚ 1994; Lin and Shih‚ 1994; Panandiker et al.‚ 1994) . Also‚ an African green monkey kidney cell line (CV-1) transfected with the enzyme peroxisomal fatty acyl-CoA oxidase. When exposed to the enzyme substrate linoleic acid for 2-6 weeks‚ 4% of the cells became tumorigenic (Chu et al.‚ 1995) .

ROS targets are many‚ including membranes and proteins. The most critical potentially carcinogenic targets of ROS are probably deoxynucleotides and DNA‚ which generate in DNA 7‚8-dihydro-8hydroxy-2'-deoxyguanine (8-OHdG)‚ 7‚8-dihydro-8-hydroxy-2'deoxyadenine (8-OHdA)‚ 2‚6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy-G)‚ and 4‚6-diamino-5-formamidopyrimidine (Fapy-A) (Bertoncini and Meneghini‚ 1995; Breen and Murphy‚ 1995; Dizdaroglu‚ 1993; Feig et al.‚ 1994; Giese et al.‚ 1995; Halliwell and Aruoma‚ 1991; Steenken‚ 1989) . Guanine is the most easily oxidized of the nucleic acid bases (an electronloss center created in a system containing the four nucleic acid bases will eventually migrate to guanine) (Steenken‚ 1989). The keto and enol forms of this damaged guanine are found in equilibrium. Thus‚ the interchangeable use of 8-oxodG and 8-OHdG found in the literature. We will use the 8-oxoG terminology in this discussion. Since the last step in formation of 8-oxoG and 8-oxoA is an oxidation while the last step in the formation of Fapy-G and Fapy-A is a reduction (Breen and Murphy‚ 1995; Steenken‚ 1989 )‚ it has been suggested that 8-oxoG formation is favored under the more oxidizing (pro-oxidant) conditions found in cancer cells (Malins et al.‚ 2003; Malins‚ 1993; Malins et al.‚ 1993) ‚ or alternatively‚ that a pro-oxidant condition exists in cells destined to become malignant favoring the accumulation of 8-oxoG. For instance‚ the DNA of hepatitis B virus infected mouse liver cells destined to become tumorigenic had elevated levels of 8-oxoG (Hagen et al.‚ 1994) . Whatever the reason‚ 8-oxoG is considered to be a major stable product generated by ROS attack on DNA and it is excreted in the urine of humans where it is used as a biological marker of in vivo oxidative DNA damage (Halliwell‚ 1993; Shigenaga et al.‚ 1994) .

The formation of 8-oxoG in DNA‚ if not repaired‚ leads to misincorporation of dA opposite to the 8-oxoG lesion. The misincorporated dA leads to an A/8-oxoG mispair resulting in G·C T· A transversions Moriya‚ 1993; Cheng et al.‚ 1992; Moriya et al.‚ 1991) . The A/8-oxo G mispair also interferes with DNA-processing enzymes such as methylases (Weitzman et al.‚ 1994) and restriction enzymes (Turk and Weitzman‚ 1995) . It is interesting to note that G·C T·A transversions occur frequently as mutations of the p53 tumor suppressor gene in human lung‚ breast‚ and liver cancers (e.g. Hollstein et al.‚ 1991) . More importantly‚ in p53‚ induces a G to T transversion at both G-residues of codon 249 (AGG) and a C to A transversion at codon 250 (CCC) (Hussain et al.‚ 1994) .

In order to minimize the deleterious effects of reactive oxygen species‚ all aerobic organisms possess extensive defense systems. The primary defense is achieved by decomposing superoxide and hydrogen peroxide‚ which thus minimizes the formation of the ensuing hydroxyl radical (·OH). This primary antioxidant defense system includes various enzymatic and nonenzymatic mechanisms such as superoxide dismutase‚ catalase‚ glutathione peroxidase‚ and ascorbic acid (e.g. Halliwell and Gutteridge‚ 1989) . In addition‚ although controversial‚ data indicate that BCL-2‚ the antiapoptotic protein also has antioxidant powers (Amstad et al.‚ 2001) . The reactive oxygen species that escape from the primary defense system have a significant chance of damaging DNA. Several repair enzymes in both prokaryotes and eukaryotes have been shown to remove 8-oxoG residues from DNA providing a secondary line of defense. The mammalian repair enzyme hOGG1‚ the human homolog of the yeast OGG1 is able to repair C/8-oxoG DNA damage back to CG in cells due to its 8-oxoG glycosylase /AP lyase activity (Gu et al.‚ 2001; Aburatani et al.‚ 1987; Arai et al.‚ 1997; Friedberg et al.‚ 1995; Hazra et al.‚ 1998; Nash et al.‚ 1996; Radicella et al.‚ 1997; Ramotar and Demple‚ 1993; Roldan-Arjona et al.‚ 1997; Shibutani et al.‚ 1991; Van der Kemp et al.‚ 1996) . The mammalian MTH1 enzyme‚ like the E. Coli MutT homolog eliminates 8-oxodGTP damage from the deoxynucleotide pool (Maki and Sekiguchi‚ 1992) . Based on sequence homology with yeast OGG1‚ hOOG1 has been cloned (Aburatani et al.‚ 1997; Arai et al.‚ 1997; Lu et al.‚ 1997; Radicella et al.‚ 1997; Roland-Arjona et al.‚ 1997; Rosenquist et al.‚ 1997; Shibutani et al.‚ 1991) . If the 8-oxoG lesion is not removed by the hOGG1 protein before DNA replication‚ then misincorporation of dA opposite to the 8-oxoG lesion occurs (Hazra et al.‚ 1998; Moriya et al.‚ 1991; Nghiem et al.‚ 1988; Shibutani et al.‚ 1991) . The mammalian hMYH enzyme‚ like E. Coli MutY‚ removes this misincorporated adenine from an A/8-oxoG mispair‚ with the formation of C/8-oxoG‚ which is then a substrate for hOGG1. This is considered the third level of defense (Au et al.‚ 1989; McGoldrick et al.‚ 1995; Slupska et al.‚ 1996; Tsai-Wu et al.‚ 1991; Yeh et al.‚ 1991) . The MutY gene has been cloned and sequenced (Boiteux et al.‚ 1987; Michaels et al.‚ 1990; Michaels et al.‚ 1991; Slupska et al.‚ 1996) . In addition‚ it has been shown that expression of the E. coli MutM gene which expresses Fpg‚ an enzyme with the same activity as hOGG1 in mammalian cells‚ reduces the mutagenicity of X-rays (Laval‚ 1994). Furthermore‚ germ line mutations in human genes hMSH2‚ or hMLH1‚ which express proteins with the ability to correct DNA mismatches and misalignments lead to genetic instability in microsatellite repeat sequences in hereditary nonpolyposis colon cancer (Jeong et al.‚ 2003; Bronner et al.‚ 1994; Papadopoulos et al.‚ 1994) ‚ It thus seems likely that the hOGG1/hMYH repair pathways are linked to protection against certain cancers.

An elevation of oxidative modifications in DNA has recently been reported in human cancers of the breast‚ prostate (Malins and Haimanot‚ 1991; Malins et al.‚ 1993‚ Malins et al.‚ 2003 and other tissues (LePage et al.‚ 2000; Olinski et al.‚ 1992) . The 8-oxoG lesion has been suggested to be a putative link to cancer development (Malins et al.‚ 2003; Malins‚ 1993 ) and a predictor for breast cancer (Malins et al.‚ 1995) . This modification occurs by the attack of hydroxyl radicals that arise from mediated by trace metal ions such as and (Luo et al.‚ 1994) . Since diffuses readily across the nuclear membrane‚ hydroxyl radicals are formed near DNA and modify it by attacking the purine and pyrimidine bases (Meneghini and Martins‚ 1993) . Breast cancer is not commonly associated with pre-existing chronic inflammatory conditions‚ unlike bowel cancer where a causal relationship between ulcerative colitis and bowel cancer has been suggested (Weitzman and Gordon‚ 1990) . Breast lesions (benign or malignant) are‚ however‚ commonly infiltrated with macrophages‚ which lead to oxidative stress. This in turn could lead to the progression of some cells from benign to malignant and/or facilitate the transformation to a more aggressive metastatic tumor. There are a number of tumorigenic cell lines including one from breast that chronically elaborate large quantities of (Szatrowski and Nathan‚ 1991) that can lead to chromosome mutations and cancer (Emerit‚ 1994) . In addition‚ the most lethal form of locally advanced breast cancer is termed inflammatory breast cancer due to the acute inflammatory changes observed in patients with this type of malignancy (Jaiyesimi et al.‚ 1992) . As mentioned above‚ humans are exposed to considerable oxidative stress as judged by measurements of up to in urine (Long et al.‚ 1999a‚b) . These levels vary with a normal diet. Halliwell concluded that urine contains "autooxidizable proteins" which upon exposure to oxygen result in the generation of to generate (Long et al.‚ 1999a) . Exposing cells to can shift the cell to a prooxidant state‚ which facilitates ROS damage to a variety of targets. We have shown this to be true in the benign human breast epithelial cell line MCF-10A (Gu et al.‚ 2001) . MCF-10A is a spontaneously immortalized human breast cell line‚ which has characteristics of human breast epithelium such as: lack of tumorigenicity in nude mice and lack of anchorage independent growth (Soule et al.‚ 1990) . We treated these cells with 5 separate time after allowing for growth (P5 line) or gradually at increments until reaching (GP line). The peroxide decay and the concomitant production of OH radicals was measured by EPR using DMPO as mentioned earlier in this chapter (Figure 1 ). After exposure to hydrogen peroxide‚ the cells became resistant to this oxidant and went on to exhibit different degrees of some of them being able to grow in immunodeficient mice (GP line). This transformation towards malignancy can be related to the cell's ability to bypass apoptosis to ensure survival‚ cell cycle anomalies modulated by oxidative stress-induceable genes and the lack of repair of 8-oxoG in DNA (Gu et al.‚ 2001) . With respect to the latter damage‚ the role of hOGG1 and hMYH repair enzymes in mammalian tumor progression is to maintain genetic stability by avoiding G . C T . A transversions. This is based on the work on E. coli MutY and MutM gene mutants. Cells with a single mutation in these genes are moderate mutators; however‚ cells with a double mutation in MutY and MutM genes have mutation rates three orders of magnitude higher than the wild-type cells Cabrera et al.‚ 1988) . Overexpression of these genes will reduce the transversion mutations in tumor promoter genes or tumor suppressor genes and thus reduce the risk of tumorigenesis. Thus‚ if indeed the role of 8-oxoG in breast cancer tumorigenesis is established‚ one application to the treatment of cancer would be elevating the enzymes that repair such lesion over the endogenous levels present in healthy tissue.

With respect to cell responses to we found that induced p53 in MCF-10A but not in the line P5. In addition‚ all transformed lines overexpress BCL-2 when compared to MCF-10A (data not shown). It has been recently shown that the transfection of MCF-10A cells with the BCL-2 gene resulted in a 5 fold increase in BCL-2 protein expression over the parental MCF-10A line (Upadhyay et al.‚ 1995) and that apoptosis did not occur in the transfectants at concentrations of In contrast‚ this concentration of induced apoptosis in the parental non-transfected line (Upadhyay et al.‚ 1995) . This apoptosis in MCF-10A cells is p53 dependent (Upadhyay et al.‚ 1995) .

The other contributors to this book have presented excellent approaches and examples to the use of EPR for observing radical species in biological systems. However‚ there are many further potential applications that currently cannot be done because of limited radical concentration or stability. This section describes alternate approaches to overcoming some of these obstacles‚ combining the two magnetic modalities that Jim Hyde has used so productively‚ often in combination with the use of spin traps. Spin traps have the potential advantages of being able to accumulate ("trap") highly reactive radicals that could not be otherwise observed directly‚ due to sensitivity limits and kinetic considerations. This is clearly the case for the hydroxyl radical‚ which may be directly observed only by fast freeze quenching at liquid nitrogen temperatures and quickly undergo secondary radical reactions and interconversions. Obviously‚ quickly immersing a mouse into liquid nitrogen negates an in vivo experiment! Many of the applications‚ successes and pitfalls of in vivo spin trapping are reviewed in this volume by Mason (ch. 5) . A major problem that is difficult to overcome is the tendency of the living cell to reduce radicals; consequently‚ the nitroxyl product of a nitrone radical adduct is normally bioreduced to the diamagnetic hydroxylamine. This section discusses potential alternatives utilizing NMR as the ultimate spectroscopic probing method. It also considers the use of more conventional NMR techniques to further characterize the spin traps and their metabolism.

Many years ago Selinsky et al. (1989) suggested the use of NMR to study free radical reactions with fluorinated spin traps. They monitored organic free radical reactions in the test tube by both EPR and NMR. In later work outlined below‚ this technique was coined as "NMR spin trapping." The success of this approach depends on the following:

1. Do the diamagnetic (decomposition or transformed) products of a spin trap adduct accumulate to sufficient levels for detection by NMR?

2. Does the resultant NMR spectrum allow extrapolation back to a specific radical adduct?

The definition encompasses not only the spin trap chemistry described below‚ but also "MRI spin trapping" described in the last section below‚ in which the weighted NMR images of water protons are measured in the presence of stable‚ paramagnetic‚ biologically generated radical adducts or complexes. The enhanced relaxation of the water protons at the site of the radicals effectively 'localizes' the site of radical generation.

Tordo and coworkers (Frejaville et al.‚ 1995) described a series of nitrone spin traps‚ based initially on DEPMPO (5-diethoxyphosphoryl-5-methyl-1pyrroline-N-oxide)‚ a phosphorus containing analog of DMPO (2‚2dimethyl-pyrroline-N-oxide). The advantages were that oxygen radical adducts of this trap showed a somewhat longer half life compared with DMPO. Despite these improvements‚ the in-vivo "stability" of paramagnetic spin trap adducts is sharply reduced relative to stable nitroxyl radicals‚ probably due to biological reduction of the resulting nitroxide to the hydroxylamine‚ but perhaps also by other destructive chemical processes.

It seems prudent to take advantage of the phosphorus moiety of DEPMPO by utilizing to probe its chemistry during and after radical reactions to understand the pathways of its radical adduct degradation. As described below, a series of biologically relevant DEPMPO adduct reaction products for and as well as methyl radical and hydroxymethyl radical were characterized by Figure 2 and ESR spectra of and reaction products. a) spectra obtained from 0.1 M DEPMPO in 0.1 M cacodylate buffer, 20 mM DTPA, pH 7.0, 10% DMSO, detected 2 h after addition of 5 mM and 5 mM at T=300K. Inset: corresponding ESR spectra observed at 10 min; b) the same reactions but in the presence of 10% methanol instead of DMSO. Inset: corresponding ESR spectra observed at 10 min.. Experimental spectra were in a good agreement with simulations using the calculated parameters for and for adducts, which are in agreement with literature data. From Khramtsov et al. (1999) with permission. concentration calculated from the integral intensity of the peak at 27.05 ppm; filled circles: the same integral intensities of the peak multiplied by 2.0 to reflect the mechanism in Scheme B. The bent curve was calculated proposing bimolecular decay of DEPMPO-OH from initial concentration equaling the concentration of Fenton reagents with the rate constant (namely where t=10 min. was the time at which the EPR spectra were obtained). The straight line was inserted as a guide to evaluate how closely the experimental data reflected a 1:1 stoichiometry between hydroxyl radical produced and product formed. Note that both the ESR data (at concentrations lower than 50 mM) and the adjusted NMR data show a linear concentration dependence on the Fenton concentration reagents. From Khramtsov et al. (1999) with permission.

The reaction of DEPMPO with radical, as shown in Scheme A, emphasizes the fact that two stereoisomers are obtained since the spin trap contains an asymmetric center (yielding, in effect, two pairs of EPR spectra). However, the radical signals decay within two hours, yielding the NMR spectra depicted in Figure 2 . For example, in Figure 2a there is evidence of several new, unique peaks at 24.54 ppm with additional peaks at 30.83 and 32.52 ppm. (A small resonance at 27.05 ppm also occurs since the reaction conditions involve Fenton chemistry (Khramtsov et al., 1999) .

These peaks originate from bimolecular disproportionation chemistry as outlined in Scheme B, where both reaction products were diamagnetic: a new nitrone (NA) and the hydroxylamine of the spin adducts, and This accounts for the three NMR peaks, one of which is 50% total intensity, two of which are 25% each (the ratio depends slightly on the stereospecificity of the initial radical addition). Since the concentrations of spin trap adduct are significantly lower in vivo, disproportionation would not be appreciable; however, direct reduction of the DEPMPO adduct to the hydroxylamines and is highly facile. Hence, when ascorbic acid was added to the radical reaction system, direct conversion to and occurred. Consequently the two new lines at 32.31 and 30.83 ppm reflected the direct "history" of spin adduct formation of the methyl radical adduct of DEPMPO and its subsequent disproportionation or in vivo bioreduction.

At high concentrations the bimolecular decay of the adduct spectrum reaction is relatively rapid. The adduct remains for only a few minutes unless balanced by extremely high steady state levels of new radical production Timmins et al., 1999) . As shown in Figure 3 , the maximum intensity of the new line at 27.05 ppm never exceeded 50% of the theoretical yield of the diamagnetic product from an adduct. Scheme C suggests that the nitrone product of disproportionation (depicted in Scheme B) is in tautomeric equilibrium with the more stable N-hydroxypyrrolidone, NA-OH, to which the NMR line at 27.05 ppm was attributed. In addition, as shown in the scheme, the hydroxylamine can spontaneously eliminate regenerating DEPMPO. Similar results were found with superoxide radical where, in fact, the NMR peaks were identical to those observed after reaction with suggesting a conversion of to (or transformation of their diamagnetic products). In vivo, however, this is unlikely to be important because the local concentrations of spin adduct are unlikely to high.

From a sensitivity viewpoint, nitrones that contain trifluoromethyl groups might be more promising than those containing other stable isotopes. This was borne out with new spin trap, FDMPO (4-hydroxy-5,5-dimethyl-2trifluoro methylpyrroline-1-oxide). The parent nitrone yields a single NMR resonance at -66.0 ppm, which yields a net ten-fold higher sensitivity than with NMR of DEPMPO. Figure 4 shows simulated and observed EPR spectra for the and adducts, respectively (Khramtsov et al., 2001) . The EPR signals were stable for over a day since bimolecular decay (as noted in Scheme B) is much less probable with the absence of an alpha proton. In order to observe the products by NMR, it was necessary to reduce the paramagnetic adduct(s) with ascorbic acid. However, despite the significantly increased in vitro stability of the paramagnetic adducts, it is likely that bioreduction would occur fairly rapidly. Figure 5 depicts results for the adduct of FDMPO in a system containing ascorbate. The inset shows the precursor EPR spectrum. spectra of adduct products: (a) in a Fenton "iron recycling" system in 0.1 M K-phosphate buffer, pH 7.0, 2 mM DTPA, 20 mM 50 mM FDPMPO, 10% v/v of DMSO, 20 mM of ascorbate, 30 min after addition of 0.5 mM FeSO4 solution; number of scans, 208; inset: kinetics of product accumulation. The symbols and denote integral intensities of the 19P-NMR peaks of reduced products at -78.6 ppm and-80.0 ppm, respectively; (b) 19F-NMR spectra of 3 mM of synthesized in Scheme B, in 0.1 M phosphate buffer, pH 7.0, before and after addition of 10 mM ascorbate; number of scans, 120; inset: EPR spectrum of 0.1 mM adduct; spectrometer settings were microwave power 20 mW; modulation amplitude, 0.63 G; sweep time, 1 min. From Khramtsov et al. (2001) with permission.

The adduct gave identical NMR spectra to that with However, overall, the fluorine containing spin traps are less likely to undergo destructive metabolism assuming toxicity levels will not be a major problem. Overall the reactivity of FDMPO was comparable to that of DMPO as measured by competitive kinetics. Consequently, the trifluoromethyl NMR spin traps have greater potential than traps containing other stable isotopes.

The remaining biological isotope, is at ca 1% natural abundance. In order to distinguish a particular molecule of interest, one must synthesize it enhanced with In a novel application by Bose-Bsau et al. (2001) .

(MNP) was employed, in the presence of as an NMR spin trap for tyrosyl radical in met-myoglobin. Relatively stable paramagnetic adducts were detectable by EPR; however, the direct identification and characterization of the final adduct on the protein and the specific chemistry involved was possible only after reduction of the paramagnetic adduct by ascorbate and the use of 2D high resolution NMR to assign the relevant Tyr residue and the chemistry of the modification. Previous spin trapping studies on myoglobin had shown the presence of tyrosyl radicals that could be trapped with MNP (Barr et al., 1996) , but the EPR results were ambiguous as to which residue was preferentially labeled. These recent results were definitive in identifying Tyr 103 in equine myoglobin and that the adduct formation occurs at the C-3 carbon of the amino acid. Figure 6 depicts the reaction scheme that most likely occurs. In addition, the studies identified the nearby protons of Phe 106. 

The direct localization and imaging of free radical distributions is much more challenging with EPR due both to aspects of stability discussed above as well as linewidth limitations. Despite almost three orders of magnitude higher sensitivity (eg, a free electron vs a proton) the much broader linewidths usually encountered in EPR places limitations on both resolution and overall sensitivity. There are additionally other limits to distinguishing tissue boundaries with radical distributions; these are simply more difficult to visualize by EPR. On the other hand, MRI, which is a mature, commercialized technology, offers exquisite anatomical resolution based on the protons of water (i.e. 100 M). Several physical aspects, such as nuclear relaxation times, diffusion rates, oxygen magnetic susceptibility effects, etc., allow multifaceted approaches to EPR and MRI image resolution. On the other hand, paramagnetic compounds serve as excellent contrast agents in MRI by inducing proton (nuclear) relaxation enhancement from rapidly exchanging water molecules. Consequently, MRI spin trapping potentially allows visualization of free radical biology at the site of its generation, by using the spin adducts as "contrast agents".

NO may be 'spin trapped' with dithiocarbamate compounds and Fe(II), which form a stoichiometric 2:1 complex for axial coordination of NO. The water soluble trap, MGD, forms the NO complex where a diagnostic EPR spectrum is found with septic shock mice (which has been almost the sole example with sufficient sensitivity in vivo). Recently Kaneko et al. (2002) demonstrated in vivo observations of NO in the brain of epileptic mice models. The relaxivity of this complex is relatively frequency independent for both and at 20 and 85 MHz, respectively. Uncomplexed MGD, Fe(II) or show essentially negligible relaxivity while the NO complex yields appreciable relaxation enhancement (Fujii et al., 1999) . Consequently, in MRI measurements, only the NO complex will yield image contrast enhancement, frequently at the site of NO generation. As shown in Figure 7 , 1.5T MRI images of septic-shock rats showed increased enhancement in the liver with time. This was suppressed by injecting the animals with a specific iNOS inhibitor, L-NMMA. Consequently, both the localization and "biochemistry" of NO generation can be visualized at high-resolution in vivo. Figure 7 . Transverse MR images of the liver in LPS-doped rats. MR images were measured at the times indicated for two different slices of the liver. Six hours after LPS injection, the NO spin-trap (3 ml of MGD: 100 mM, Fe: 20 mM) was administered i.p. The slice thickness was 2 mm, and each slice was separated by 1 mm. The image enhancement was drastically reduced upon treatment with the NOS inhibitor, L-NMMA. Adapted from Fujii et al. (1999) with permission.

Most of the new approaches outlined here are still in their infancy. The future, however, looks promising Feasibility studies have been quite successful. Most of the stable isotopes incorporated in the spin traps described above are easily resolvable in the presence of other biological milieu. Sensitivity is particularly promising with NMR, a technique that has never been exploited to its full capacity in vivo.

Of particular note is the use of paramagnetic relaxation enhanced MRI. The power of paramagnetic contrast agents to MRI has been proven time and again. Several sophisticated pulse sequences exist which take advantage of small susceptibility/relaxation effects. In addition, the development of new, targeted paramagnetic spin traps and spin probes promise to provide the requisite localization. The ability to observe biological radical production at the site of generation offers the ultimate advantage in diagnosis.

There clearly are many areas in which free radicals are intrinsically involved in both physiology and pathophysiology. There also are many situations where the involvement of free radicals is minimal or not significant. EPR spectroscopy, combined with thoughtful experimental approaches can be a powerful method for resolving many of the questions that arise regarding the role of free radicals in disease. The National Biomedical ESR Center at MCW has had, and will continue to have an important role in providing critical EPR spectroscopy tools and original experimental results that will provide an understanding of the role of free radicals in disease. There appear to be sufficient important problems to make this a productive field for many years.

Cloning and characterization of mammalian 8-hydroxyguaninespecific DNA glycosylase/apurinic, apyrimidinic lyase, a functional mutM homologue

Oxidants are a major contributor to cancer and aging

Oxidants, antioxidants, and the degenerative diseases of aging

Separation of oxidant-initiated and redox-regulated steps in the signal transduction pathway

Cloning of a human homolog of the yeast OGG1 gene that is involved in the repair of oxidative DNA damage

BCL-2 is involved in preventing oxidant-induced cell death and in decreasing oxygen radical production

Oxygen radical inhibition of nitric oxide-dependent vascular function in sickle cell disease

Escherichia coli mutY gene encodes an adenine glycosylase active on G/A mispairs

The histochemistry of lipofuscin and neuromelanin

ESR spin trapping of a protein-derived tyrosyl radical from the reaction of cytochrome c with hydrogen peroxide

Apoptosis and its role in human disease

The role of oxidative stress in the pathogenesis of age-related macular degeneration

Phospholipid peroxidation in tumor promoter-exposed mouse skin

DNA strand breaks produced by oxidative stress in mammalian cells exhibit 3'-phosphoglycolate termini

Formamidopyrimidine-DNA glycosylase of Escherichia coli: cloning and sequencing of the fpg structural gene and overproduction of the protein

Electron spin resonance investigations of human retinal pigment epithelium melanosomes from young and old donors

The Pigmentary System. Physiology and Patophysiology

Ocular pathology in mice with a transgenic insertion at the microphthalmia locus

Protein NMR spin trapping with application to the tyrosyl radical of equine myoglobin Free Rad

Acute pulmonary injury in rats by nitrofurantoin and modification by vitamin E, dietary fat, and oxygen

Reactions of oxyl radicals with DNA

Mutation in the DNA mismatch repair gene homologue hMLHI is associated with hereditary nonpolyposis colon cancer

Amyotrophic lateral sclerosis: recent insights from genetics and transgenic mice

SOD1 aggregates in ALS: cause, correlate, or consequence

Superoxide and hydrogen peroxide in relation to mammalian cell proliferation

Redox regulation of programmed cell death in lymphocytes

Oxidative stress as a mediator of apoptosis

Elevated basal reactive oxygen species and phospho-Akt in murine keratinocytes resistant to ultraviolet B-induced apoptosis

) mutM, a second mutator locus in Escherichia coli that generates transversions

Oxidative damage and protection of the RPE

Dopamine neurotoxicity: agedependent behavioral and histological effects

The Respiratory burst oxidase

8-hydroxyguanine, an abundant form of oxidative DNA damage causes G T and A C substitutions

Monochromosome transfer provides functional evidence for growth-suppressive genes on chromosome 14 in nasopharyngeal carcinoma

Transformation of mammalian cells by overexpressing peroxisomal fatty acyl-CoA oxidase

Growth and pigmentation in genetically related S91 Cloudman melanoma cell lines treated with IBMX and MSH

Monoamine oxidase and oxidative stress at dopaminergic synapses

The pathobiology of Parkinson's disease: biochemical aspects of dopamine neuron senescence

Apoptosis: molecular control point in toxicity

Oxidant stress induces the protooncogenes c-fos and c-myc in mouse epidermal cells

Sunlight and age-related macular degeneration. The beaver dam eye study

Fos: An immediate-early transcription factor in neurons

The Bcl-2 family of proteins, and the regulation of neuronal survival

Principles and Practice of Infectious Diseases

Reduction of paraquat and related bipyridylium compounds to free radical metabolites by rat hepatocytes

Basal lipid peroxidation in substantia nigra is increased in Parkinson's disease

Chemistry of free radical damage to DNA and nucleoproteins

Reactive oxygen species, chromosome mutation, and cancer: Possible role of clastogenic factors in carcinogenesis. Free Rad

A standardized test for the identification and characterization of melanins using electron paramagnetic resonance (EPR) spectroscopy

Purified human neuromelanin, synthetic dopamine melanin as a potential model pigment, and the normal human substantia nigra: characterization by electron paramagnetic resonance spectroscopy

The roles of neuromelanin, binding of metal ions, and oxidative cytotoxicity in the pathogenesis of Parkinson's disease: a hypothesis

Is the incidence of registrable age-related macular degeneration increasing? Brit

Reverse chemical mutagenesis: Identification of the mutagenic lesions resulting from reactive oxygen species-mediated damage to DNA

Spin trapping of superoxide and hydroxyl radical: practical aspects

Oxygen-regulated control elements in the phosphoglycerate kinase 1 and lactate dehydrogenase A genes: Similarities with the erythropoietin 3' enhancer

Oxidative stress-induced apoptosis prevented by trolox. Free Rad

DNA repair

DNA Repair and Mutagenesis

(Diethoxyphosphoryl)-5-methyl-1-pyrroline -oxide: a new efficient phosphorylated nitrone for the in vitro and in vivo spin trapping of oxygen-centered radicals

In vivo EPR studies of the metabolic fate of nitrosobenzene in the mouse

In vivo imaging of spin-trapped nitric oxide in rats with septic shock: MRI spin trapping

In vivo evidence of free radical formation after asbestos instillation: an ESR spin trapping investigation. Free Rad

The chemistry of single-stranded 4'-DNA radicals: influence of the radical precursor on anaerobic and aerobic strand cleavage

Cell damage in inflammatory and infectious sites might involve a coordinated "cross-talk" among oxidants, microbial haemolysins and ampiphiles, cationic proteins, phospholipases, fatty acids, proteinases and cytokines

Mossbauer spectroscopic studies of purified human neuromelanin isolated from the substantia nigra

Detection of nitrosyl hemoglobin in venous blood in the treatment of sickle cell anemia with hydroxyurea

Tobacco smoke tumor promoters, catechol and hydroquinone, induce oxidative regulation of protein kinase C and influence invasion and metastasis of lung carcinoma cells

Bicarbonate enhances the peroxidase activity of Cu, Zn-superoxide dismutase

Oxidative stress and apoptosis in HIV infection: a role for plant-derived metabolites with synergistic antioxidant activity

Catecholamine toxicity: a proposal for the molecular pathogenesis of manganese neurotoxicity and Parkinson's disease

Mutant Cu, Zn,-superoxide dismutase in motor neuron disease

Alteration of DNA base excision repair enzymes hMYH and hOGG1 in hydrogen peroxide resistant transformed human breast cells

Tyrosinase and inhibition of proliferation of melanoma cells and fibroblasts

Oxidative DNA damage: meaning and measurement

DNA damage by oxygen-derived species. Its mechanism and measurement in mammalian systems

Free Radicals in Biology and Medicine

Extensive oxidative DNA damage in hepatocytes of transgenic mice with chronic active hepatitis destined to develop hepatocellular carcinoma

The nature of retinal radiation damage: dependence on wavelength, power level and exposure time

The presence of two distinct 8-oxoguanine repair enzymes in human cells: their potential complementary roles in preventing mutation

Spontaneous oxygen radical generation by sickle erythrocytes

Comparative action spectrum for ultraviolet light killing of mouse melanocytes from different genetic coat color backgrounds

bcl-2, a novel regulator of cell death

The Interaction of bovine erythrocyte superoxide dismutase with hydrogen peroxide: inactivation of the enzyme

Mutations in human cancers

Paraquat uptake into freshly isolated rabbit lung epithelial cells and its reduction to the paraquat radical under anaerobic conditions

Redox-active chalcogen-containing glutathione peroxidase mimetics and antioxidants inhibit tumor promoter-induced downregulation of gap junctional intercellular communication between WB-F344 liver epithelial cells

Iron nitrosyl hemoglobin formation from the reactions of hemoglobin and hydroxyurea

Oxy-radical induced mutagenesis of hotspot codons 248 and 249 of the human p53 gene

Signal integration at the c-fos promoter

Inflammatory Breass Cancer: A Review (1992)

Iron-melanin complex in substantia nigra of parkinsonian brains:an X-ray microanalysis

Oxidative stress as a cause of Parkinson's disease

Microsatellite instability and mutations in DNA mismatch repair genes in sporadic colorectal cancer

In vivo evidence of free radical formation in the rat lung after exposure to an emission source air pollution particle

Consequences of nitric oxide generation in epileptic-seizure rodent models as studied by in vivo EPR

Is the vulnerability of neurons in the substantia nigra of patients with Parkinson's disease related to their neuromelanin content?

Electron spin resonance imaging of mouse B16 melanoma

Application of the EPR spin trapping technique to the detection of radicals produced in vivo during inhalation exposure of rats to ozone

Measurements of in vivo parameters pertinent to ROS/RNS using EPR spectroscopy

Reactive oxygen species as cellular messengers

Spin traps: in vitro toxicity and stability of readical adducts. Free Rad

NMR Spin Trapping: detection of free radical adducts using a phosphorus containing nitrone spin trap

NMR spin trapping: detection of free radical reactions using a new fluorinated DMPO analog, Free Rad

Role of free radicals in the pathogenesis of acute chest syndrome in sickle cell disease

Prevalence of age-related maculopathy. The beaver dam eye Study

Hypoxia causes the activation of nuclear factor through the phosphorylation of on tyrosine residues

Hypoxia Activation of nuclear factor is mediated by a Ras and Raf signaling pathway and does not involve MAP Kinase (ERK1 or ERK2)

Antioxidant action of neuromelanin: the inhibitory effect on lipid peroxidation

Genetic Recombination

The ORD1 gene encodes a transcription factor involved in oxygen regulation and is identical to IXR1, a gene that confers cisplatin sensitivity to Saccharomyces cerevisiae

Interaction between chemicals and melanin

Macrophages and inflammatory mediators in tissue injury

Expression of the E. coli fpg gene in mammalian cells reduces the mutagenicity of xrays

Transcription coupled repair of 8-oxoguanine: requirement for XPG, TFIIH, and CSB and implications for Cockayne syndrom

On the role of bicarbonate in peroxidations catalyzed by Cu, Zn, superoxide dismutase. Free Rad

Inhibitory effect of curcumin on xanthine dehydrogenase/oxidase induced by phorbol-12-myristate-13-acetate in NIH3T3 cells

Increased mitochondrial antioxidative activity or decreased oxygen free radical propagation prevent mutant SODl-mediated motor neuron cell death and increased amyotrophic lateral sclerosis-like transgenic mouse survival

Evaluation of DEPMPO as a spin trapping agent in biological systems. Free Rad

Gd(3+) and Yb(3+) induced changes in mitochondrial structure, membrane permeability, cytochrome c release and intracellular ROS level

Mutator phenotype may be required for multistage carcinogenesis

Microsatellite instability: marker of a mutator phenotype in cancer

Hydrogen peroxide in human urine: implications for antioxidant defense and redox regulation

Generation of hydrogen peroxide by "antioxidant" beverages and the effect of milk addition. Is cocoa the best beverage? Free Radio

Electron spin resonance studies on living cells. III. Development of single individuals in Triturus alpestris

Three chemically distinct types of oxidants formed by iron-mediated Fenton reactions in the presence of DNA

A mammalian DNA repair enzyme that excises oxidatively damaged oxoguanine maps to a locus frequently lost in lung cancer

In vivo detection of anthralin-derived free radicals in the skin of hairless mice by low-frequency electron paramagnetic resonance spectroscopy

MutT protein specifically hydrolyzes a potent mutagenic substrate for DNA synthesis

Identification of hydroxyl radical-induced lesions in DNA as structure biomarkers with a putative link to cancer development

Major alterations in the nucleotide structure of DNA in cancer of the female breast

The etiology of breast cancer: Characteristic alterations in hydroxyl radical-induced DNA bas lesions during oncogenesis with potential for evaluating incidence risk

Cancer-related changes in prostate DNA as men age early identification of metastasis in primary prostate tumors

The Etiology and Prediction of Breast Cancer

The Development of the Human Eye

Modulation of oxidant formation in mouse skin in vivo by tumor-promoting phorbol esters

Pheomelanin as a binding site for drugs and chemicals

Phosphorylated nitroxides in the pyrrolidine series: reduction by ascorbate. Free Radic

Free radical metabolites of foreign compounds and their texicological significance

Free radical intermediates in the metabolism of toxic chemicals

The mechanism of microsomal and mitochondrial nitroreductase. Electron spin resonance evidence for nitroaromatic free radical intermediates

The role of catalytic superoxide formation in the inhibition of nitroreductase

Light and risk for age-related eye diseases

Characterization of a mammalian homolog of the Escherichia coli

Hydrogen peroxide and DNA damage

MutM, a protein that prevents G:C to T:A transversions, is formamidopyrimidine-DNA glycosylase

MutY, an adenine glycosylase active on G:A mispairs, has homology to endonuclease III

Single-stranded shuttle phagemid for mutagenesis studies in mammalian cells: 8-Oxoguanine in DNA induces targeted G-C transversions in simian kidney cells

Site-specific mutagenesis using a gapped duplex vector: a study of translesion synthesis past 8-oxodeoxyguanosine in E. coli

Absence of ferritin protein in substantia nigra pars compacta neurons. A reappraisal to the role of iron in Parkinson disease pathogenesis

Direct detection of free radical generation in an in vivo model of acute lung injury

Induction of reactive oxygen intermediates in human monocytes by tumor cells and their role in spontaneous monocyte cytotoxicity

Cloning of yeast 8-oxoguanine DNA glycosylase reveals the existence of a base-excision DNA-repair protein superfamily

Antioxidant status and free radicalinduced oxidative damage of sickle erythrocytes

The mutY gene: a mutator locus in Escherichia coli that generates G:C to T:A transversions

Vertebrate craniofacial development: the relation between ontogenic process and morphological outcome

Transcriptional activation of early-response genes by hydrogen peroxide in a mouse osteoblastic cell line

The clonal evolution of tumor cell populations

DNA base modifications in chromatin of human cancerous tissues

Neuromelanin of the human substantia nigra: a mixed-type melanin

Genetic-, age-and light-induced degeneration of the retina and retinal pigment epithelium

The Biogenesis of Melanosomes

Dose-response effects of malachite green on free radical formation, lipid peroxidation and DNA damage in Syrian hamster embryo cells and their modulation by antioxidants

Mutation of a mutL homolog in hereditary colon cancer

Age-related macular degeneration. 1. Epidemiology, pathogenesis and differential diagnosis

Chapter 28. The Enzymology of melanogenesis

6-Dihydroxyindole is a melanin precursor showing potent cytotoxicity

Effect of selenium and vitamin E deficiency on nitrofurantoin toxicity in the chick

Studies on skin aging. Preparation and properties of fucose-rich oligo-and polysaccharides. Effect on fibroblast proliferation and survival

) c-fos proto-oncogene regulation and function

Nitrone spin traps and their pyrrolidine analogs in myocardial reperfusion injury: hemodynamic and ESR implications

Quantitative determination of melanin in pigmented cells by electron spin resonance spectroscopy

Transplantable melanomas in gerbils (Meriones unguiculatus). II -melanogenesis

Motor neuron disease and animal models

Biological effects of cigarette smoke, wood smoke, and the smoke from plastics: the use of electron spin resonance. Free Rad

Widespread bimodal intrachromosomal genomic instability in sporadic breast cancers associated with 13q allelic imbalance

Cloning and characterization of hOGG1, a human homolog of the OGG1 gene of saccharomyces cerevisiae

Enzymes that repair oxidative damage to DNA

Generation of radical anions of nitrofurantoin, misonidazole, and metronidazole by ascorbate

Generation of nitro radical anions of some 5-nitrofurans, and 2-and 5-nitroimidazoles by rat hepatocytes

Chapter 32. Retinal phototoxicity

The retinal pigmented epithelium is required for development and maintenance of the mouse neural retina

Cell-free hemoglobin limits nitric oxide bioavailability in sicklecell disease

Low levels of reactive oxygen species as modulators of cell function

How many mutations are required for tumorgenesis? Implications from human cancer data

Hydroxyl radical formation by sickle erythrocyte membranes: role of pathologic iron deposits and cytoplasmic reducing agents

Transition metals, ferritin, glutathione, and ascorbic acid in parkinsonian brains

Molecular cloning and functional expression of a human cDNA encoding the antimutator enzyme 8-hydroxyguanine-DNA glycosylase

Tissue uptake of paraquat and diquat

Cloning and characterization of a mammalian 8-oxoguanine DNA glycosylase

Quantitative measurement of superoxide generation using the spin trap 5-(Diethoxyphoshporyl)-5-methyl-1-pyrroline-N-oxide

Photoreactivity of aged human RPE melanosomes: a comparison with lipofuscin

OR7 -Photooxidation of retinal pigment melanin -a model for melanin aging

Free radical scavenging properties of melanin interaction of eu-and pheo-melanin models with reducing and oxidising radicals. Free Rad

Inhibition of activation-induced death in T cell hybridoma by thiol antioxidants: oxidative stress as a mediator of apoptosis

Properties and function of the ocular melanin--a photobiophysical view

Interactions of melanin with oxygen (and related species)

Loss of melanin in human RPE with aging

Photobiology of the Eye

Retinal melanin and lipofuscin: possible role in photoprotection and phototoxicity of human RPE

In vivo lipid-derived free radical formation by NADPH oxidase in acute lung injury induced by lipopolysaccharide: a model for ARDS

Generation of superoxide anion and hydrogen peroxide by erythrocytes from individuals with sickle trait or normal hemoglobin

Reactive oxygen intermediates as apparently widely used messengers in the activation of the transcription factor and HIV-1

Development of fluorinated, NMR-active spin traps for studies of free radical chemistry

Comparative oxidation of hemoglobins A and S

Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG

Assays of oxidative DNA damage biomarkers 8-oxo-2'-deoxyguanosine and 8-oxoguanine in nuclear DNA and biological fluids by high-performance liquid chromatography with electrochemical detection methods

Binding of iron to neuromelanin of human substantia nigra and synthetic melanin: an EPR spectroscopy study. Free Rad

Reexamination of the mechanism of hydroxyl radical adducts formed from the reaction between familial amyotrophic lateral sclerosis-associated Cu,Zn superoxide dismutase mutants and H2O2

Cloning and sequencing a humal homolog (hMYH) of the Escherichia coli MutY gene whose function is required for the repair of oxidative DNA damage

Reduced and oxidized glutathione in the substantia nigra of patients with Parkinson's disease

Isolation and Characterization of a Spontaneously Immortalized Human Breast Epithelial Cell Line, MCF-10

Human tumor suppressor genes

Purine bases, nucleosides, and nucleotides: aqueous solution redox chemistry and transformation reactions of their radical cations and and OH adducts

Free radicals in cancer

Use of electron spin resonance spectroscopy to study cancer

Potential medical (clinical) applications of EPR: Overviews and perspectives in In Vivo EPR(ESR): Theory and Applications

Modulation by neuromelanin of the availability and reactivity of metal ions

Production of large amounts of hydrogen peroxide by human tumor cells

Visual disabilities of oculocutaneous albinism and their alleviation

The long-term effects of visible light on the eye

Trapping of free radicals with direct in vivo EPR detection: a comparison of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) as spin traps for and Free Rad

Nucleotide sequence of the Escherichia coli micA gene required for A/G-specific mismatch repair: Identity of MicA and MutY

Free radical DNA adduct 8-OH-deoxyguanosine affects activity of HPA II and MSP 1 restriction endonucleases

) bcl-2 suppresses expression of in breast epithelial cells

Cloning and expression of Escherichia coli of the OGG1 gene of saccharomyces cerevisial, which codes for a DNA glycosidose that excises 7,8-dihidro-8-oxoguanine and 2,6-diamino-4-hydroxy-5-N-methylformamido pyrimidine

Spectroscopic studies of substantia nigra pigment in human subjects

Heme oxygenase 1 mediates an adaptive response to oxidative stress in human skin fibroblasts

Oncogenes, antioncogenes, and the molecular basis of multistep carcinogenesis

Relationship of senile macular degeneration to ocular pigmentation

Inflamation and cancer: role of phagocyte-generated oxidants in carcinogenesis

Free radical adducts induce alterations in DNA cytosine methylation

Altered reactivity of superoxide dismutase in familial amyotrophic lateral sclerosis

Albino spatial vision as an instance of arrested visual development

What's the use of generating melanin?

An intron splice acceptor polymorphism in hMSH2 and risk of leukemia after treatment with chemotherapeutic agents

Analysis of c-Fos and c-Jun redox-dependent DNA binding activity

The redox and DNA-repair activities of Ref-1 are encoded by nonoverlapping domains

Measurement of free radicals from smoke inhalation and oxygen exposure by spin trapping and ESR spectroscopy

Activation of AP-1 and of a nuclear redox factor, REF-1, in the response of HT29 colon cancer cells to hypoxia

Two nicking enzyme systems specific for mismatch-containing DNA in nuclear extracts from human cells

Solar radiation and age-related macular degeneration

Age-related macular degeneration: review of pathogenesis

The effect of a synthetic neuromelanin on yield of free hydroxyl radicals generated in model systems

Total and paramagnetic metal ions in human substantia nigra and its neuromelanin

The neuromelanin of human substantia nigra and its interaction with metals

Bicarbonate enhances the hydroxylation, nitration, and peroxidation reactions catalyzed by copper, zinc superoxide dismutase

Bicarbonate enhances peroxidase activity of Cu, Zn-superoxide dismutase-Role of carbonate anion radical and scavenging of carbonate anion radical by metalloporphyrin antioxidant enzyme mimetics