Robert F. Anderson

Energetics of the one-electron reduction steps of riboflavin, FMN and FAD to their fully reduced forms

Abstract The one-electron reduction potentials ( E 1 7 ) of riboflavin, FMN and FAD have been determined using pulse radiolysis from the position of the electron-transfer equilibria between flavins and reference quinones in aqueous solution. The average value for all three flavins E 1 7 (F/FH . ) = − 314 ± 8 mV is used to calculate the second-electron reduction potential of the flavins E 2 7 (FH . /FH 2 (FH − )) = − 124 mV.

Energetics of the one-electron steps in the NAD+/NADH redox couple

The one-electron reduction potential of NAD+ has been determined using pulse radiolysis to study electron-transfer equilibria between it and a low potential bipyridylium compound. The determined value E1 electron pH7 (NAD+/NAD) = -922 +/- 8 mV (NHE scale) is used to calculate E2 electrons pH7 (NAD/NADH) = +282 mV. E1 electron pH7 for 1-methylnicotinamide, E1 electron pH7 (MeN+/MeN) = -918 +/- 7 mV.

Hypoxia-selective radiosensitization of mammalian cells by nitracrine, an electron-affinic DNA intercalator.

The radiosensitizing ability of the 1-nitroacridine nitracrine (NC) is of interest since it is an example of a DNA intercalating agent with an electron-affinic nitro group. NC radiosensitization was evaluated in Chinese hamster ovary cell (AA8) cultures at 4 degrees C in order to suppress the rapid metabolism and potent cytotoxicity of the drug. Under hypoxic conditions, submicromolar concentrations of NC resulted in sensitization (SER = 1.6 at 1 mumol dm-3). Sensitization was also seen under aerobic conditions but a concentration more than 10-fold higher was required. In aerobic cultures NC radiosensitization was independent of whether cells were exposed before and during, or after, irradiation. Postirradiation sensitization was not observed under hypoxic conditions. The time dependence of NC uptake and the development of radiosensitization were similar (maximal at 30 min at 4 degrees C under hypoxia) suggesting that sensitization, unlike cytotoxicity, is due to unmetabolized drug. NC is about 1700 times more potent as a radiosensitizer than misonidazole. This high potency is adequately accounted for by the electron affinity of NC (E(1) value at pH7 of -275 mV versus NHE) and by its accumulation in cells to give intracellular concentrations approximately 30 times greater than in the medium. However, concentrations of free NC appear to be low in AA8 cells, presumably because of DNA binding. If radiosensitization by NC is due to bound rather than free drug, it suggests that intercalated NC can interact very efficiently with DNA target radicals. This is despite a binding ratio in the cell estimated as less than 1 NC molecule/400 base pairs under conditions providing efficient sensitization. This work suggests a new approach in the search for more effective clinical radiosensitizers, and poses questions on the means by which intercalated drugs can interact with DNA damage.

Oxidative metabolism of amsacrine by the neutrophil enzyme myeloperoxidase

Oxidative metabolism of the anti-cancer drug amsacrine 4-(9-acridinylamino) methane-sulphan-m-anisidide has been suggested to account for its cytotoxicity. However, enzymes capable of oxidizing it in non-hepatic tissue have yet to be identified. A potential candidate, that may be relevant to the metabolism of amsacrine in blood and its action in myeloid leukaemias and myelosuppression, is the haem enzyme myeloperoxidase. We have found that the purified human enzyme oxidizes amsacrine to its quinone diimine, either directly or through the production of hypochlorous acid. In comparison, the 4-methyl-5-methylcarboxamide derivative of amsacrine, CI-921 9-[[2-methoxy-4[(methylsulphonyl)-amino]phenyl]amino)-N, 5-dimethyl-4-acridine carboxamide, reacted poorly with myeloperoxidase, although it was oxidized by hypochlorous acid. Detailed studies of the mechanism by which myeloperoxidase oxidizes amsacrine revealed that the semiquinone imine free radical is a likely intermediate in this reaction. Oxidation of amsacrine analogues indicated that factors other than their reduction potential determine how readily they are metabolized by myeloperoxidase. Both amsacrine and CI-921 inhibited production of hypochlorous acid by myeloperoxidase. CI-921 acted by trapping the enzyme as the inactive redox intermediate compound II. Amsacrine inhibited by a different mechanism that may involve conversion of myeloperoxidase to compound III, which is also unable to oxidize Cl-. The susceptibility of amsacrine to oxidation by myeloperoxidase indicates that this reaction may contribute to the cytotoxicity of amsacrine toward neutrophils, monocytes and their precursors.

Reduction of nitroimidazoles in model chemical and biological systems

Some chemical and biological properties of intermediates obtained during reduction of nitroimidazoles are discussed. These include: rate data for the decay of the nitro radical-anion, stoichiometry and absorption spectra for reduction via the radical-anion or using dithionite, stoichiometry with other reducing agents, and rate of reduction by xanthine/xanthine oxidase. Increased radiosensitization by misonidazole is seen upon prolonged pre-irradiation incubation using E. coli, enabling demonstration that a freely-diffusable metabolite is responsible for this effect. Preliminary experiments designed to extend studies of the radiobiological properties of extracellularly-added metabolites to mammalian cells and the use of liver perfusion to generate metabolites are described.

The application of wide band transformers to the study of transient conductivity in pulse irradiated aqueous solutions by the D.C. method

A new pulse radiolysis conductivity technique is described, in which a transformer, constructed from coaxial cable wound on a toroidal magnetic core, is used to overcome the problems associated with the high intrinsic conductance of aqueous solutions. In one form, the transformer is wound in a balanced-to-unbalanced configuration so that the effects of the primary electrons stopped in the conductivity cell electrodes are eliminated, thus enabling fast time resolution to be obtained. This type of transformer may be used to study transient species with lifetimes of between 50 ns and 5 miros. A reversing transformer which may be used for the investigation of species with longer lifetimes (5 micros-1 ms) is also described. The method is suitable for use with aqueous solutions containing up to 5 × 10-3 mol dm-3 of ion pairs. The application of these transformers and their associated electronics is described and illustrated with examples of their use. The mathematical relationships between the observed voltage signal and the conductance change in the solution are derived for the systems described.

THE EFFECT OF 1,4‐DIAZABICYCLO[2.2.2]OCTANE ON THE RADIOSENSITIVITY OF BACTERIA

Abstract— Hydroxyl radicals (OH) are scavenged by 1,4‐diazabicyclo[2.2.2]octane (DABCO) at a diffusion‐controlled rate of 1.25 ± 0.1 × 109M‐1s‐1. Unlike other efficient OH scavengers which exhibit protection of bacteria against irradiation both in oxic and hypoxic conditions, DABCO has been shown to protect Serratia marcescens and various strains of Escherichia coli only in oxic conditions.

The radiation-chemical yields of H3O+ and OH- as determined by nanosecond conductimetric measurements☆

Abstract The radiation-chemical yields of ionic species formed upon irradiation of water by 3.5 MeV electrons have been determined directly using dc conductivity and optical measurements. Yields (expressed in μmol J-1) at 10 and 110 ns after the end of a 10 ns pulse are: for H3O+ = 0.371, 0.320; for OH- = 0.082, 0.045, and for e-aq = 0.299 and 0.275, respectively.

The bimolecular decay rates of the flavosemiquinones of riboflavin, FMN and FAD

The bimolecular decay rates (2k) of the flavosemiquinones (FH·F⨪) of riboflavin, FMN and FAD have been determined using pulse radiolysis. The rates (defined as d[FH·F⨪]dt = −2k[FH·F⨪]2) for the neutral flavosemiquinones at zero ionic strength and pH 5.9 are (in units of mol−1·dm3·s−1): (1.2 ± 0.1)·109, (5.0 ± 0.2)·108 and (1.4 ± 0.1)·108; and for the anionic flavosemiquinones at pH 11.2 (5.4 ± 0.9)·108, (4.5 ± 0.3)·107 and (8.5 ± 1.3)·106, respectively. The kinetic salt effect has been used to formulate rate equations for each flavin to adjust for ionic strength effects.

Oxidation of the cyclohexadienyl radical by metal ions: A pulse radiolysis study

Abstract The rates of oxidation of the cyclohexadienyl radical by metal ions in water have been measured. The bimolecular rate constants (dm 3 mol -1 s -1 ) and corresponding reduction potentials ( E 0 ) were: Fe(C 2 O 4 ) 3- 3 : (1.30 ± 0.05) × 10 6 (0.002 V); Cu(H 2 O) 2+ 6 : (3.6 ± 0.6) × 10 6 (0.167 V); Fe(H 2 O) 3+ 6 : (2.5 ± 0.4) × 10 7 (0.77 V) and Ce 4+ · n H 2 O: (1.5 ± 0.1) × 10 8 (1.70 V). Oxidation of the cyclohexadienyl radical by V(H 2 O) 3+ 6 (-0.25 V) and Cr(H 2 O) 3+ 6 (-0.40 V) ions was not observed ( k 5 dm 3 mol -1 s -1 ) and it is suggested that the reduction potential of the cyclohexadienyl radical lies between -0.25 V and 0 V.