Eric D. Clarke

Oxygen inhibition of nitroreductase: Electron transfer from nitro radical-anions to oxygen

Abstract The inhibition of many nitroreductases by oxygen has been explained by Mason and Holtzman in terms of electron transfer to oxygen from the nitro radical-anions, which have been identified as the first intermediate in some reductase systems. We have used the pulse radiolysis technique to measure the bimolecular rate constants of this electron-transfer reaction for over 20 nitro compounds, including substituted 2- and 5-nitroimidazoles of interest as antiprotozoal drugs and radiosensitizers, nitrofurans in use as antibacterial agents, and substituted nitrobenzenes previously used as model substrates for nitroreductases. The logarithm of the rate constant for the reaction of the nitro radical-anion with oxygen is linearly related to the one-electron reduction potential of the nitro compound.

Mammalian cell toxicity of nitro compounds: Dependence upon reduction potentiakl

Summary The toxicity of several classes of nitro-aromatic and -heterocyclic compounds towards W79 mammalian cells in vitro has been determined. Cells with varying concentrations of drugs were incubated in air at 37°C for up to 14 days in order to form colonies. It was found that the concentration of nitro compound required to reduce cell colony-forming ability by 50% was a function of the one-electron reduction potential of the compound, more cytotoxic compounds having more positive potentials.

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

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.

Nitroimidazoles as anaerobic electron acceptors for xanthine oxidase

The rates of anaerobic nitroreduction of 15 nitroimidazoles by xanthine-xanthine oxidase were measured. The compounds studied were mainly 2-nitroimidazoles, including misonidazole and analogues of potential value in cancer therapy; most 5-nitroimidazoles such as metronidazole and nimorazole reacted too slowly for measurement under the conditions used. Using 0.1 mM nitroimidazole, reduction rates varied between ca. 4 and 680 nmole min-1 U-1 xanthine oxidase at 37 degrees and pH 7.4. These rates were correlated with the reduction potential characterizing one-electron reduction to the nitro radical-anion, and compared with those for other nitroaryl compounds and with reduction by free flavin mononucleotide.

Nitroaryl compounds as potential fluorescent probes for hypoxia. I. Chemical criteria and constraints

Cellular reduction of nitroaryl compounds is efficiently inhibited by oxygen, and detection of products characteristic of reduction could form the basis for diagnostic tests for the presence of hypoxic cells in tumors. The criteria for suitable compounds include a high sensitivity and selectivity of detection response between oxic and hypoxic cells, which can be provided using fluorescence detection and suitable nitroaryl compounds which have very low fluorescence until reduced. Examples described include a nitroacridine and nitronaphthalimides. Although the intercalating ability of these ring systems lead to high sensitivity for detection of reduced metabolites in vitro by flow cytometry, poor bioavailability is an unwanted consequence of intercalation. The application of several model reducing systems for reduction of potential fluorescent probes for hypoxia is described, and the absorption and fluorescence spectral characteristics of other examples of structures which could form the basis for useful probes are outlined.

Nitroaryl compounds as potential fluorescent probes for hypoxia. II. Identification and properties of reductive metabolites

Nitroakridin 3582 (NA) and a nitronaphthalimide (DM113), which fluoresce only upon reduction, have been studied by HPLC. V79-379A cells incubated with NA under 20% or 2% O2 and N2 gave increasing amounts of the fluorescent amine with an hypoxic:oxic differential of 160. Measurement of the uptake of NA showed that it was concentrated within the cell by over 1000-fold. Studies in 3 different cell lines of reduction under hypoxia showed a 7-fold range in amine production. DM113 yields more than one fluorescent product, which show different absorption and fluorescence spectra. Chemical reduction of NA or DM113 using a variety of methods gave, depending on conditions, amine and/or (what was presumed to be) hydroxylamine; the latter was non-fluorescent. In vivo, NA is toxic at greater than 0.19 mumol g-1. At this dose much of the drug is found in the liver and kidneys. Plasma levels at 30 minutes are only 2 microM while tumor concentrations are 10 microM compared to 600 microM in the liver. However, the half life is greater than 1 hr and amine was detectable in these tumors.

Cis-trans isomerization of the (5-nitro-2-furyl) acrylamide, AF-2, initiated by ascorbate, glutathione, Fe(II) and OH−

The cis-trans isomerization of the (5-nitro-2-furyl)acrylamide, AF-2, has been investigated using some important biological reducing agents to initiate reaction. Physiological concentrations of L-ascorbic acid, glutathione and iron(II) all accomplish isomerization in a catalytic manner over a period of minutes. Base-catalysed isomerization has also been observed. In all cases, the presence of oxygen severely inhibits isomerization. It is proposed that the mechanism involves a free-radical chain process; AF-2 or analogues are thus extremely sensitive probes for the generation of nitro radicals in biochemical reducing systems because of the high efficiency of isomerization.

The mechanism of the free-radical-induced chain isomerisation of 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

The cis-trans isomerisation of 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) has been studied by pulse and steady-state radiolysis techniques. A free-radical chain-isomerisation mechanism is proposed. Initiation involves production of nitro radical-anions from cis-AF-2 with subsequent isomerisation to give the trans-AF-2 radical. Propagation of the chain reaction occurs via electron transfer between the isomers and their radical-anions, disproportionation of the radicals representing the main chainterminating step. Oxygen acts as an efficient inhibitor of isomerisation by electron-transfer reaction with the nitro radical-anions. Computer simulations using the Gear numerical integration algorithm show this mechanism to match the observed results closely.

Temperature Effects on the One-Electron Reduction Potentials of Nitroaryl Compounds

Pulse radiolysis was used to establish one-electron transfer equilibria between radical cations of methyl or benzyl viologens (V2+) and nitroaryl compounds (ArNO2): a nitroimidazole (misonidazole or metronidazole), 4-nitrobenzoate or nitrofurazone. The equilibrium constants in water at pH 8 were estimated over the temperature range approximately 5 to 75 degrees C. The difference delta E in mid-point one-electron reduction potentials between the nitro compounds and the viologens varied with temperature T; increasing temperature made the nitro compounds apparently less electron-affinic compared to the effects of temperature on the viologen potential. Values of delta(delta E)/delta T were in the range -0.7 to -1.1 mV K-1 at 25 degrees C. If delta[E(V2+/V.+)]/delta T = -0.9 mV K-1 for methyl viologen then delta[E(ArNO2/ArNO2.-)]/delta T is about -2 mV K-1 for these compounds.

Electron-Transfer Reactions of Nitroaromatic Radical-Anions Observed by Pulse Radiolysis

Nitroaromatic radical-anions react with oxygen to produce superoxide ion in water at pH~8. The rate constants of this one-electron transfer reaction, measured by pulse radiolysis, were 1 to 4 orders of magnitude below the diffusion-controlled limit. Measurements of the accompanying free-energy changes (from the equilibrium constants of reactions with redox indicators) revealed a linear free-energy relationship between the logarithm of the rate constant and ΔG°. The Eyring activation energy ΔG≠ was also dependent upon ΔG°.