Radiolysis Studies of Oxidation and Nitration of Tyrosine and Some Other Biological Targets by Peroxynitrite-Derived Radicals.

The widespread interest in free radicals in biology extends far beyond the effects of ionizing radiation, with recent attention largely focusing on reactions of free radicals derived from peroxynitrite (i.e., hydroxyl, nitrogen dioxide, and carbonate radicals). These radicals can easily be generated individually by reactions of radiolytically-produced radicals in aqueous solutions and their reactions can be monitored either in real time or by analysis of products. This review first describes the general principles of selective radical generation by radiolysis, the yields of individual species, the advantages and limitations of either pulsed or continuous radiolysis, and the quantitation of oxidizing power of radicals by electrode potentials. Some key reactions of peroxynitrite-derived radicals with potential biological targets are then discussed, including the characterization of reactions of tyrosine with a model alkoxyl radical, reactions of tyrosyl radicals with nitric oxide, and routes to nitrotyrosine formation. This is followed by a brief outline of studies involving the reactions of peroxynitrite-derived radicals with lipoic acid/dihydrolipoic acid, hydrogen sulphide, and the metal chelator desferrioxamine. For biological diagnostic probes such as 'spin traps' to be used with confidence, their reactivities with radical species have to be characterized, and the application of radiolysis methods in this context is also illustrated.

Calculation of Standard Reduction Potentials of Amino Acid Radicals and the Effects of Water and Incorporation into Peptides.

Guanine (Guo) is generally accepted as the most easily oxidized DNA base when cells are subjected to ionizing radiation; calculations of the standard reduction potential of the guanyl radical, Eo(Guo•+/Guo) are within ∼0.1 V of experimental values in aqueous solution extrapolated to standard conditions. While a number of experimental studies have shown some amino acid radicals have redox properties at pH 7 which suggest or confirm a capacity for radical "repair" by electron transfer from the amino acid to Guo•+ (or its deprotonated conjugate), the redox properties of the radicals of other amino acids, including methionine, lysine and cystine, are less well characterized. In addition, the effects of incorporation of the amino acids into peptides, or the effects of water of hydration on calculated potentials, have not been extensively studied. In this work, calculations of standard reduction potentials of radicals from model amino acids as they appear in histone proteins are performed. To predict redox properties at pH 7, acid dissociation constants (pKas) of both radical and ground state amino acids are required. In some instances these are not experimentally determined and calculated pKas have been derived for some common amino acids and compared with experimental values.

Calculations of the Energetics of Oxidation of Aqueous Nucleosides and the Effects of Prototropic Equilibria.

Recently the calculated standard reduction potentials of the radical-cations of N-methyl substituted DNA bases have been reported that agree fairly well with the experimental results. However, there are issues reflecting the fact that the experimental results usually relate to the couple E(o)(Nuc(•),H(+)/NucH(+)), whereas the calculated results are for the E(o)(Nuc(•+)/Nuc) couple. To calculate the midpoint reduction potential at pH 7 (Em7), it is important to have accurate acid dissociation constants (pKs) for both ground-state bases and their radicals, and the effects of uncertainty in some of these values (e.g., that of the adenosine radical) must be considered. Calculations of the pKs of the radicals of the nucleic acid bases (as nucleosides) have been performed to explore the effects the various pKs have on calculating the values of Em7 and to see what improvements can be made with the accuracy of the calculations.

Radiation-Chemical Perspective of the Radiobiology of Pulsed (High Dose-Rate) Radiation (FLASH): A Postscript.

An earlier commentary (Wardman P, Radiat Res. 2020; 194:607-617) discussed possible chemical reaction pathways that might be involved in the differential responses of tissues to high- vs. low-dose-rate irradiation, focusing on reactions between radicals, and radiolytic depletion of a chemical influencing radiosensitivity. This brief postscript updates discussion to consider recent modeling and experimental studies, and presents more detail to support the earlier suggestion that rapid depletion of nitric oxide will certainly occur after a radiation pulse of a few grays, underlining the need to include the consequences of such a change when considering FLASH effects.

Factors Important in the Use of Fluorescent or Luminescent Probes and Other Chemical Reagents to Measure Oxidative and Radical Stress.

Numerous chemical probes have been used to measure or image oxidative, nitrosative and related stress induced by free radicals in biology and biochemistry. In many instances, the chemical pathways involved are reasonably well understood. However, the rate constants for key reactions involved are often not yet characterized, and thus it is difficult to ensure the measurements reflect the flux of oxidant/radical species and are not influenced by competing factors. Key questions frequently unanswered are whether the reagents are used under 'saturating' conditions, how specific probes are for particular radicals or oxidants and the extent of the involvement of competing reactions (e.g., with thiols, ascorbate and other antioxidants). The commonest-used probe for 'reactive oxygen species' in biology actually generates superoxide radicals in producing the measured product in aerobic systems. This review emphasizes the need to understand reaction pathways and in particular to quantify the kinetic parameters of key reactions, as well as measure the intracellular levels and localization of probes, if such reagents are to be used with confidence.

Approaches to modeling chemical reaction pathways in radiobiology.

PURPOSE: Recent interest in understanding effects of high dose-rate ('FLASH') radiobiology has prompted a number of groups to model the chemical reactions that might be involved, either to estimate radiolytic oxygen consumption in tissues, or the yields and persistence of specific reactive intermediates or products. However, most models have been either not biomimetic and/or inadequately supported by kinetic data. This review summarizes issues which should be addressed in developing models for chemical reactions in radiobiology. CONCLUSIONS: A model should be based on mechanistic pathways that lead to well-defined chemical and biological endpoints: crucially, the pathways should be plausibly similar in both the model and cells or tissues, and reflect the Law of Mass Action. Complex calculations of radiolytic yields are unnecessary, as reasonable estimates based on experimental data are generally available. Different parts of the intracellular milieu (such as the cytoplasm, nucleus, or phospholipid membranes) should be addressed separately, or with two-compartment models where appropriate. Homogeneous kinetics can be used as a first step in modeling, but the heterogeneity - both of radiolytic damage distribution and of cellular reactants - will need to be addressed. Major problems arise in choosing appropriate rate constants and estimating intracellular concentrations of reactants in the different organelles. It helps to identify and focus on the key reactions, as complex models may mask deficiencies and/or uncertainties; but it is still important to include all reactions and reactants that can have a significant effect on the model, as well as build upon experience in modeling chemical pathways in biology.

Kinetics of reduction of tyrosine phenoxyl radicals by glutathione.

Modification of tyrosine (TyrOH) is used as a marker of oxidative and nitrosative stress. 3,3'-Dityrosine formation, in particular, reflects oxidative damage and results from the combination of two tyrosyl phenoxyl radicals (TyrO·). This reaction is in competition with reductive processes in the cell which 'repair' tyrosyl radicals: possible reductants include thiols and ascorbate. In this study, a rate constant of 2 x 106 M⁻¹ s⁻¹ was estimated for the reaction between tyrosyl radicals and glutathione (GSH) at pH 7.15, generating the radicals by pulse radiolysis and monitoring the tyrosyl radical by kinetic spectrophotometry. Earlier measurements have suggested that this 'repair' reaction could be an equilibrium, and to investigate this possibility the reduction (electrode) potential of the (TyrO·,H+/TyrOH) couple was reinvestigated by observing the fast redox equilibrium with the indicator 2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonate). Extrapolation of the reduction potential of TyrO· measured at pH 9-11 indicated the mid-point reduction potential of the tyrosyl radical at pH 7, E(m7)(TyrO·,H+/TyrOH) = 0.93 ± 0.02 V. This is close to the reported reduction potential of the glutathione thiyl radical, E(m7) = 0.94 ± 0.03V, confirming the 'repair' equilibrium constant is of the order of unity and suggesting that efficient reduction of TyrO· by GSH might require removal of thiyl radicals to move the equilibrium in the direction of repair. Loss of thiyl radicals, facilitating repair of TyrO·, can arise either via conjugation of thiyl with thiol/thiolate or oxygen, or unimolecular transformation, the latter important at low concentrations of thiols and oxygen.

The effects of nitric oxide or oxygen on the stable products formed from the tyrosine phenoxyl radical.

Tyrosine is a critical component of many proteins and can be the subject of oxidative posttranslational modifications. Furthermore, the oxidation of tyrosine residues to phenoxyl radicals, sometimes quite stable, is essential for some enzymatic functions. The lifetime and fate of tyrosine phenoxyl radicals in biological systems are largely driven by the availability and proximity of oxidants and reductants. Tyrosine phenoxyl radicals have extremely low reactivity with molecular oxygen whereas reactions with nitric oxide are diffusion controlled. This is in contrast to equivalent reactions with tryptophanyl and cysteinyl radicals where reactions with oxygen are much faster. Despite, the quite disparate apparent reactivity of tyrosine phenoxyl radicals with oxygen and nitric oxide being known, the products of the reactions are not well established. Changes in the levels from expected basal concentrations of stable products resulting from tyrosine phenoxyl radicals, for example naturally occurring 3,3'-dityrosine, 3-nitrotyrosine, and 3-hydroxytyrosine, can be indicative of oxidative and/or nitrosative stress. Using the radiolytic generation of specific oxidizing radicals to form tyrosine phenoxyl radicals in an aqueous solution at a known rate, we have compared the products in the absence and presence of nitric oxide or oxygen. Possible reactions of the phenoxyl radicals with oxygen remain unclear although we show evidence for a small decrease in the yield of dityrosine and loss of tyrosine in the presence of 20% oxygen. Low concentrations of nitric oxide in anoxic conditions react with tyrosine phenoxyl radicals, by what is most probably through the formation of an unstable intermediate, regenerating tyrosine and forming nitrite.

Radiotherapy Using High-Intensity Pulsed Radiation Beams (FLASH): A Radiation-Chemical Perspective.

Radiation chemists have been routinely using high-dose microsecond-pulsed irradiation for almost 60 years, involving many thousands of studies, in the technique of "pulse radiolysis". This involves dose rates broadly similar to the FLASH regimen now attracting interest in radiotherapy and radiobiology. Using the experience gained from radiation chemistry, two scenarios are examined here that may provide a mechanistic basis for any differential response in normal tissues versus tumors in FLASH radiotherapy. These are: 1. possible depletion of a chemical critical to the response to radiation, and 2. radical-radical reactions as a possible cause of effects occurring mainly with high-intensity pulsed radiation. The evidence for changes in relative levels of so-called "reactive oxygen species" produced after irradiation using FLASH versus conventional irradiation modalities is also examined.

Kinetics of reaction of nitrogen dioxide with dihydrorhodamine and the reaction of the dihydrorhodamine radical with oxygen: implications for quantifying peroxynitrite formation in cells.

Dihydrorhodamine 123 (RhH2) has been used to detect 'reactive nitrogen species', including peroxynitrite and its radical decomposition products, peroxynitrite probably oxidizing RhH2 to rhodamine (Rh) via radical products rather than directly. In this study, the radical intermediate (RhH(.)) was generated by pulse radiolysis, and shown to react with oxygen with a rate constant k approximately 7 x 10(8) M(-1) s(-1). This fast reaction was exploited in experiments observing Rh being formed slowly (k approximately 4-7 x 10(5) M(-1) s(-1)) from oxidation of RhH2 by nitrogen dioxide in a rate-limiting step, >1000-fold slower than the corresponding oxidation by carbonate radicals. The time-dependent uptake of RhH2 into mammalian cells was measured, with average intracellular levels reaching only approximately 10 microM with the protocol used. The combination of low loading and relatively low reactivity of oxidants towards RhH2 compared to competing cellular nucleophiles suggests rather a small fraction of peroxynitrite-derived radicals (mainly CO3(.-)) may be scavenged intracellularly by RhH2.

Reaction of hydrated electrons with guanine derivatives: tautomerism of intermediate species.

Here, we show that two tautomers are produced by the protonation of the guanine-electron adduct. The fate of electron adducts of a variety of substituted guanosines was investigated by radiolytic methods and addressed computationally by means of time-dependent DFT (TD-B3LYP/6-311G**//B1B95/6-31+G**) calculations. The reaction of e(aq-) with guanosine and 1-methylguanosine produces two transient species, whereas the reaction with N2-ethylguanosine and N2,N2-diethylguanosine produces only one. The two short-lived intermediates, which show a substantial difference in their UV-visible spectra, are recognized to be two purine tautomers (i.e., iminic 18 and aminic 19 forms). The tautomerization 18 --> 19 occurs with a rate constant of ca. 1.5 x 106 s(-1) , and theory suggests that it is a water-assisted process.

Radiation chemistry comes before radiation biology.

PURPOSE: This article seeks to illustrate some contributions of radiation chemistry to radiobiology and related science, and to draw attention to examples where radiation chemistry is central to our knowledge of specific aspects. Radiation chemistry is a mature branch of radiation science which is continually evolving and finding wider applications. This is particularly apparent in the study of the roles of free radicals in biology generally, and radiation biology specifically. The chemical viewpoint helps unite the spatial and temporal insight coming from radiation physics with the diversity of biological responses. While historically, the main application of radiation chemistry of relevance to radiation biology has been investigations of the free-radical processes leading to radiation-induced DNA damage and its chemical characterization, two features of radiation chemistry point to its wider importance. First, its emphasis on quantification and characterization at the molecular level helps provide links between DNA damage, biochemical repair processes, and mutagenicity and radiosensitivity. Second, its central pillar of chemical kinetics aids understanding of the roles of 'reactive oxygen species' in cell signalling and diverse biological effects more generally, and application of radiation chemistry in the development of drugs to enhance radiotherapy and as hypoxia-specific cytotoxins or diagnostic agents. The illustrations of the broader applications of radiation chemistry in this article focus on their relevance to radiation biology and demonstrate the importance of synergy in the radiation sciences. CONCLUSIONS: The past contributions of radiation chemistry to radiation biology are evident, but there remains considerable potential to help advance future biological understanding using the knowledge and techniques of radiation chemistry.

Nitroimidazoles as hypoxic cell radiosensitizers and hypoxia probes: misonidazole, myths and mistakes.

Nitroimidazoles have been extensively explored as hypoxic cell radiosensitizers but have had limited clinical success, with efficacy restricted by toxicity. However, they have proven clinically useful as probes for tumour hypoxia. Both applications, and probably much of the dose-limiting toxicities, reflect the dominant chemical property of electron affinity or ease of reduction, associated with the nitro substituent in an aromatic structure. This single dominant property affords unusual, indeed extraordinary flexibility in drug or probe design, suggesting further development is possible in spite of earlier limitations, in particular building on the benefit of hindsight and an appreciation of errors made in earlier studies. The most notable errors were: the delay in viewing cellular thiol depletion as a likely common artefact in testing in vitro; slow recognition of pH-driven concentration gradients when compounds were weak acids and bases; and a failure to explore the possible involvement of pH and ascorbate in influencing hypoxia probe binding. The experience points to the need to involve a wider range of expertise than that historically involved in many laboratories when studying the effects of chemicals on radiation response or using diagnostic probes.

The roles of thiol-derived radicals in the use of 2',7'-dichlorodihydrofluorescein as a probe for oxidative stress.

2',7'-Dichlorodihydrofluorescein (DCFH2) is one of the most widely used probes for detecting intracellular oxidative stress, but requires a catalyst to be oxidized by hydrogen peroxide or superoxide and reacts nonspecifically with oxidizing radicals. Thiyl radicals are produced when many radicals are "repaired" by thiols, but are oxidizing agents and thus potentially capable of oxidizing DCFH2. The aim of this study was to investigate the reactivity of thiol-derived radicals toward DCFH2 and its oxidized, fluorescent form 2',7'-dichlorofluorescein (DCF). Thiyl radicals derived from oxidation of glutathione (GSH) or cysteine (CysSH) oxidized DCFH2 with rate constants at pH 7.4 of approximately 4 or approximately 2x10(7) M(-1) s(-1), respectively. Both the rates of oxidation and the yields of DCF were pH-dependent. Glutathione-derived radicals interacted with DCF, resulting in the formation of DCFH* absorbing at 390 nm and loss of fluorescence; in contrast, cysteine-derived radicals did not cause any depletion of DCF fluorescence. We postulate that the observed apparent difference in reactivity between GS* and CysS* toward DCF is related to the formation of carbon-centered, reducing radicals from base-catalyzed isomerization of GS*. DCF formation from interaction of DCFH2 with GS* was inhibited by oxygen in a concentration-dependent manner over the physiological range. These data indicate that in applying DCFH2 to measure oxidizing radicals in biological systems, we have to consider not only the initial competition between thiols and DCFH2 for the oxidizing radicals, but also subsequent reactions of thiol-derived radicals, together with variables--including pH and oxygen concentration--which control thiyl radical chemistry.

Thiyl radicals react with nitric oxide to form S-nitrosothiols with rate constants near the diffusion-controlled limit.

A possible route to S-nitrosothiols in biology is the reaction between thiyl radicals and nitric oxide. D. Hofstetter et al. (Biochem. Biophys. Res. Commun.360:146-148; 2007) claimed an upper limit of (2.8+/-0.6)x10(7) M(-1)s(-1) for the rate constant between thiyl radicals derived from glutathione and nitric oxide, and it was suggested that under physiological conditions S-nitrosation via this route is negligible. In the present study, thiyl radicals were generated by pulse radiolysis, and the rate constants of their reactions with nitric oxide were determined by kinetic competition with the oxidizable dyes 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) and a phenothiazine. The rate constants for the reaction of nitric oxide with thiyl radicals derived from glutathione, cysteine, and penicillamine were all in the range (2-3) x10(9) M(-1)s(-1), two orders of magnitude higher than the previously reported estimate in the case of glutathione. Absorbance changes on reaction of thiyl radicals with nitric oxide were consistent with such high reactivity and showed the formation of S-nitrosothiols, which was also confirmed in the case of glutathione by HPLC/MS. These rate constants imply that formation of S-nitrosothiols in biological systems from the combination of thiyl radicals with nitric oxide is much more likely than claimed by Hofstetter et al.

Methods to measure the reactivity of peroxynitrite-derived oxidants toward reduced fluoresceins and rhodamines.

The commonest probes for "reactive oxygen and nitrogen species" are reduced fluorescein and rhodamine dyes that fluoresce when oxidized. The reduced dyes are reactive toward peroxynitrite, although probably not directly but via free radical oxidants derived from it: hydroxyl, carbonate, and nitrogen dioxide free radicals. The reaction with peroxynitrite can be monitored by rapid mixing and stopped-flow spectrophotometry, but reliable measurement of reactivity of the peroxynitrite-derived radicals requires specialized techniques such as flash photolysis or pulse radiolysis to monitor the fast reactions in real time. A key feature of oxidation by radicals is that the reaction produces an intermediate fluorescein or rhodamine radical, which normally is oxidized further by oxygen to yield the fluorescent, stable product. Susceptibility of the yield of fluorescence to interference by antioxidants can be assessed from kinetic parameters, which reflect reactivity. This chapter outlines methods for estimation of key rate constants involving peroxynitrite-derived oxidants.

Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects.

Chemical probes for free radicals in biology are important tools; fluorescence and chemiluminescence offer high detection sensitivity. This article reviews progress in the development of probes for "reactive oxygen and nitrogen" species, emphasizing the caution needed in their use. Reactive species include hydrogen peroxide; hydroxyl, superoxide, and thiyl radicals; carbonate radical-anion; and nitric oxide, nitrogen dioxide, and peroxynitrite. Probes based on reduced dyes lack selectivity and may require a catalyst for reaction: despite these drawbacks, dichlorodihydrofluorescein and dihydrorhodamine have been used in well over 2,000 studies. Use in cellular systems requires loading into cells, and minimizing leakage. Reactive species can compete with intracellular antioxidants, changes in fluorescence or luminescence possibly reflecting changes in competing antioxidants rather than free radical generation rate. Products being measured can react further with radicals, and intermediate probe radicals are often reactive toward antioxidants and especially oxygen, to generate superoxide. Common probes for superoxide and nitric oxide require activation to a reactive intermediate; activation is not achieved by the radical of interest and the response is thus additionally sensitive to this first step. Rational use of probes requires understanding and quantitation of the mechanistic pathways involved, and of environmental factors such as oxygen and pH. We can build on this framework of knowledge in evaluating new probes.

Reaction of the carbonate radical with the spin-trap 5,5-dimethyl-1-pyrroline-N-oxide in chemical and cellular systems: pulse radiolysis, electron paramagnetic resonance, and kinetic-competition studies.

Carbonate radicals (CO3-) can be formed biologically by the reaction of OH with bicarbonate, the decomposition of the peroxynitrite-carbon dioxide adduct (ONOOCO2-), and enzymatic activities, i.e., peroxidase activity of CuZnSOD and xanthine oxidase turnover in the presence of bicarbonate. It has been reported that the spin-trap DMPO reacts with CO3(-) to yield transient species to yield finally the DMPO-OH spin adduct. In this study, the kinetics of reaction of CO3(-) with DMPO were studied by pulse radiolysis, yielding a second-order rate constant of 2.5 x 10(6) M(-1) s(-1). A Fenton system, composed of Fe(II)-DTPA plus H2O2, generated OH that was trapped by DMPO; the presence of 50-500 mM bicarbonate, expected to convert OH to CO3(-), markedly inhibited DMPO-OH formation. This was demonstrated to be due mainly to a fast reaction of CO3(-) with FeII-DTPA (k=6.1 x 10(8) M(-1) s(-1)), supported by kinetic analysis. Generation of CO3(-) by the Fenton system was further proved by analysis of tyrosine oxidation products: the presence of bicarbonate caused a dose-dependent inhibition of 3,4-dihydroxiphenylalanine with a concomitant increase of 3,3'-dityrosine yields, and the presence of DMPO inhibited tyrosine oxidation, in agreement with the rate constants with OH or CO3(-). Similarly, the formation of CO3(-) by CuZnSOD/H(2)O(2)/bicarbonate and peroxynitrite-carbon dioxide was supported by DMPO hydroxylation and kinetic competition data. Finally, the reaction of CO3(-) with DMPO to yield DMPO-OH was shown in peroxynitrite-forming macrophages. In conclusion, CO3(-) reacts quite rapidly with DMPO and may contribute to DMPO-OH yields in chemical and cellular systems; in turn, the extent of oxidation of other target molecules (such as tyrosine) by CO3(-) will be sensitive to the presence of DMPO.

Radiosensitization by nitric oxide at low radiation doses.

Nitric oxide was shown to radiosensitize anoxic V79 and CHO hamster cells and MCF7 and UT-SCC-14 human cells, measuring clonogenic survival and/or DNA damage in vitro at low radiation doses (0.1-5 Gy). Radiosensitization was easily detected after 2 Gy in anoxic V79 cells exposed to 40 ppm ( approximately 70 nM) nitric oxide, indicating that nitric oxide is a significantly more efficient radiosensitizer than oxygen. The yield of double-strand breaks (as gamma-H2AX foci) in V79 and MCF7 cells was doubled by irradiation in 1% v/v nitric oxide/N(2), and there was a longer repair time in cells irradiated in nitric oxide than in air or anoxia; single-strand breaks ("comet" assay) also appeared to be enhanced. Potent radiosensitization by nitric oxide is consistent with near diffusion-controlled reaction of nitric oxide with purine and pyrimidine radicals observed by pulse radiolysis, with nitric oxide reacting two to three times faster than oxygen with the 5-hydroxy-uracil-6-yl radical. Stable NO/base adducts were formed with uracil radicals. Effects on the radiosensitivity of cells exposed to as low as 40 ppm v/v nitric oxide after doses of 1-2 Gy suggest that variations in radiosensitivity in individual patients after radiotherapy might include a component reflecting differing levels of nitric oxide in tumors.