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

Quinone chemistry and toxicity

Publisher Summary This chapter focuses on the electron transfer reactions in organic chemistry. The simplest elementary act in homogeneous solution chemistry is the exchange of an electron between two chemical entities. No bonds need to be formed or broken in the process, and if the two species are identical, except for a difference in oxidation state, no net chemical change takes place. Such self-exchange reactions constitute the basis of theoretical treatments of electron transfer. A more complex and chemically interesting situation is the electron-transfer reaction between two species of different chemical composition. In electron-transfer theory, it is an important postulate that electrons are transferred one by one, which does not mix well with the two-electron centered electronic theory of organic molecules. Organic electron transfers rarely occur as isolated steps because of the high chemical reactivity of odd-electron species. Processes involving one or several electron-transfer steps are not likely to preserve the stereochemical integrity of the reactants. With the present upsurge of research studies frequently invoking organic electron-transfer steps in mechanistic discussions, there is a great need of a theoretical background applicable to organic species and compatible with the language of physical organic chemistry.

Electron transfer reactions in organic chemistry

Free radical formation by antitumor quinones

Publisher Summary Many free radicals of biological interest are highly reactive and never reach a concentration high enough to be detected by electron paramagnetic resonance (EPR). An example of this is the hydroxyl radical, which reacts with itself or with most organic molecules at diffusion-controlled rates. Its rate of reaction is limited mainly by the frequency with which it collides with other species. Thus, the direct detection of hydroxyl radicals by EPR in a biologic system is impossible. For short-lived radicals of lesser reactivity compared to the hydroxyl radical, there are various means of detection using EPR. A simple method is to slow the rate of disappearance of the radical by rapidly freezing the sample. This has the disadvantage that the radical is no longer in a fluid environment, and the resultant anisotropic effects can obscure the identification of the radical. In theory, spin trapping can overcome many of these difficulties. This technique consists of using a spin trap––that is, a compound that forms a stable free radical by reacting covalently with an unstable free radical. This chapter discusses the spin trapping of the biologically important free radicals: superoxide and hydroxyl.

Spin trapping of superoxide and hydroxyl radicals.

Oxidation of halide, thiocyanate, carbonate and selenite ions by hydroxyl radicals yields radicalanions which themselves oxidise amino-acids such as tryptophan and tyrosine. The free-radical product(s) of oxidation of tryptophan (TrpH) by Br–2, (SCN)–2, CO–3 and a selenite free radical have similar spectral and kinetic properties in neutral and alkaline solutions. The spectral shifts and decay kinetics of the tryptophan free-radical observed at pH ∼ 3 show that oxidation of TrpH by Br–2 and (SCN)–2 gives a radical which protonates (pKa= 4.3) and which is singly charged at pH ∼ 3. The different prototropic behaviour of the absorption of the product of oxidation of TrpH by the selenite radical suggests that in this case, more than one species is produced. The protonated free radical TrpH+ reacts with SCN– so that there is an apparent loss of reactivity of (SCN)–2 towards TrpH, especially at low pH, high [SCN–] and low [TrpH]. A kinetic model for the (SCN)–2/TrpH reaction predicts a variation of the equilibrium concentration of (SCN)–2 with [H+], [TrpH] and [SCN–]. The experimental data are in accord with the model and enable the equilibrium constants of the reactions to be obtained.

Mechanism of tryptophan oxidation by some inorganic radical-anions: a pulse radiolysis study

The differences between the one-electron reduction potentials at pH 7 (E17) of a quinone couple (Q/Q–), and seventeen nitroimidazole couples (S/S–) were estimated for S = substituted 2-, 4-, and 5-nitroimidazoles, by measuring the equilibrium constant Kc of the reaction S–+ Q ⇌ S + Q–. The radical-ions S– and Q– were generated by pulse radiolysis, and their concentrations measured spectrophotometrically after the equilibrium was attained (typically 1–50 µs after the pulse). Estimated values of E17(S/S–) range from –243 mV for S = 5-formyl-1-methyl-2-nitroimidazole; –398 mV for 1-(2-hydroxyethyl)-2-nitroimidazole; –486 mV for 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole to ⩽–527 mV for 4-nitroimidazole (all ± 10 mV). In the absence of other factors, 2-nitroimidazoles have more positive E17 values than the 5-nitro-analogues, and 4-nitroimidazoles are still weaker oxidants. Substitution with electron-withdrawing groups in the side chain can increase E17 by amounts greater than the difference between 2- and 5-nitroimidazoles. These values are based on E17(Q/Q–)=–244 ± 7 mV for duroquinone, and –375 ± 8 mV for 9,10-anthraquinone-2-sulphonate. These reference potentials were determined by measuring Kc for one-electron transfer equilibria between the quinones and 1,1′-dibenzyl-4,4′-bipyridylium ion (BV2+), assuming E17(BV2+/BV+)=–354 ± 6 mV. The usefulness of viologens as redox indicators in this type of study is discussed.

One-electron reduction potentials of substituted nitroimidazoles measured by pulse radiolysis

The semiquinones, Q.-, of derivatives of 2- and 6-methyl-1,4-naphthoquinones, some incorporating leaving groups with substituents such as CH/sub 2/Br or CH/sub 2/OCONHCH/sub 3/, have been produced by radiolytic reduction of Q by (CH/sub 3/)2COH radicals. The absorption spectra and decay kinetics of Q.- were all closely similar to that produced from 2-methyl-1,4-naphthoquinone, with no evidence for unimolecular elimination of a leaving group in the semiquinone form, but immediate loss of leaving group upon two-electron reduction of Q to the hydroquinone. The redox equilibria between Q/Q.- and O2/O2.- were characterized, and reduction potentials of the couples Q/Q.- in water at pH 7.6 were calculated. The implications of these observations for the use of these compounds as bioreductive alkylating agents or as radiosensitizers with potential selective activity toward hypoxic cells are discussed.

One-electron reduction of 2- and 6-methyl-1,4-naphthoquinone bioreductive alkylating agents

Purpose: The study aimed to identify suitable prodrugs that could be used to test the hypothesis that peroxidase activity in cells, either endogenous or enhanced by immunological targeting, can activate prodrugs to cytotoxins. We hypothesized that prototype prodrugs based on derivatives of indole-3-acetic acid (IAA), when activated by peroxidase enzymes (e.g., from horseradish, HRP) should produce peroxyl radicals, with deleterious biological consequences. Methods and Materials: V79 hamster cells were incubated with IAA or derivatives 6 HRP and cytotoxicity assessed by a clonogenic assay. To assess the toxicity of stable oxidation products, prodrugs were also oxidized by HRP without cells, and the products then added to cells. Results: The combination of prodrug and enzyme resulted in cytotoxicity, but neither indole nor enzyme in isolation was toxic under the conditions used. Although lipid peroxidation was stimulated in liposomes by the prodrug/enzyme treatment, it could not be measured in mammalian cells. Adding oxidized prodrugs to cells resulted in cytotoxicity. Conclusions: Although the hypothesis that prodrugs of this type could enhance oxidative stress via lipid peroxidation was not established, the results nonetheless demonstrated oxidatively-activated cytotoxicity via indole acetic acid prodrugs, and suggested these as a new type of substrate for antibody-directed enzyme-prodrug therapy (ADEPT). The hypothesized free-radical fragmentation intermediates were demonstrated, but lipid peroxidation associated with peroxyl radical formation was unlikely to be the major route to cytotoxicity. © 1998 Elsevier Science Inc. Indole-3-acetic acids, Peroxidases, Oxidation therapy, Enzyme-prodrug therapy, ADEPT.

c Hypoxic Stress Proteins and Gene Regulation TOWARD TARGETED "OXIDATION THERAPY" OF CANCER: PEROXIDASE-CATALYSED CYTOTOXICITY OF INDOLE-3-ACETIC ACIDS

Peter Wardman

Papers

Mechanism of tryptophan oxidation by some inorganic radical-anions: a pulse radiolysis study

One-electron reduction potentials of substituted nitroimidazoles measured by pulse radiolysis

One-electron reduction of 2- and 6-methyl-1,4-naphthoquinone bioreductive alkylating agents

c Hypoxic Stress Proteins and Gene Regulation TOWARD TARGETED "OXIDATION THERAPY" OF CANCER: PEROXIDASE-CATALYSED CYTOTOXICITY OF INDOLE-3-ACETIC ACIDS

Free radicals in biology: the pulse radiolysis approach