Roger H. Bisby

Repair of amino acid radicals by a vitamin E analogue

Free radicals derived from one-electron oxidation of the amino acids tryptophan, tyrosine, methionine and histidine have been found to be rapidly (k = 10(7) -10(9) dm3 mol-1 s-1) and efficiently repaired by Trolox C, a vitamin E analogue. The reactions form a relatively stable phenoxyl radical of Trolox C (lambda max = 440 nm; epsilon = 5.4 X 10(3) mol dm-3 cm-1). The radical cation of tryptophan is more rapidly repaired than the neutral tryptophan radical. Repair of tryptophanyl radicals in the enzyme lysozyme has also been observed. The results suggest that a function of alpha-tocopherol in membranes may be the repair of radicals of integral membrane proteins.

Active Uptake of Drugs into Photosensitive Liposomes and Rapid Release on UV Photolysis

Abstract Liposomes containing high concentrations of the anticancer drug doxorubicin, prepared by active-loading techniques, have been intensively investigated as potential agents for chemotherapy. The present study investigates the possibility of active uptake and photoinduced release of such solutes from liposomes incorporating a photoisomerizable lipid. The active loading of acridine orange and doxorubicin was investigated using liposomes containing entrapped ammonium sulfate. The liposomes were prepared with dipalmitoyl-l-α-phosphatidyl choline (DPPC) and a photochromic lipid, (1,2-(4′-n-butylphenyl)azo-4′-(γ-phenylbutyroyl))-glycero-3-phosphocholine (Bis-Azo PC), which isomerizes on exposure to near-UV light with resulting changes in membrane permeability to solutes. The rate of loading of the vesicles below the phase transition temperature of DPPC was investigated as a function of Bis-Azo PC and cholesterol concentrations in the liposome. The rate of doxorubicin uptake was found to be greatly decreased in the presence of cholesterol, while below 30°C the rate of acridine orange uptake was increased in the presence of cholesterol. On exposure to a single UV laser pulse, actively loaded acridine orange was rapidly released from liposomes containing Bis-Azo PC at a rate similar to that found for the indicator dye calcein. However while cholesterol had previously been shown to greatly enhance the rate of photoinduced calcein leakage, it had no significant effect on the rate of acridine orange release. After active loading into DPPC vesicles containing Bis-Azo PC, doxorubicin was also released after exposure to a single laser pulse, but at a rate slower than for acridine orange and calcein. The difference in behavior between these systems is ascribed to the interactions of acridine orange and doxorubicin with the liposome bilayer. Photoinduced release of pharmacologically active materials from sensitized liposomes might provide a useful adjunct or alternative to conventional photodynamic therapy.

Reactions of the α-tocopheroxyl radical in micellar solutions studied by nanosecond laser flash photolysis

Laser flash photolysis of α‐tocopherol in methanol and in aqueous micellar solutions has been shown to produce the α‐tocopheroxyl radical. The reaction between the α‐tocopheroxyl radical and ascorbate in positively charged hexadecyltrimethylammonium chloride (HTAC) micelles occurred with a second order rate constant of 7.2 × 107 M−1·s−1, whereas in negatively charged sodium dodecyl sulphate (SDS) micelles the rats constant was only 3.8 × 104 M−1·s−1. The α‐tocopheroxyl radical was found to be relatively long‐lived in HTAC micelles (t½ ⩾ 5 min), allowing the slow disappearance of the α‐tocopheroxyl radical by reaction with glutathione to be observed.

Properties of the radicals formed by one-electron oxidation of acetaminophen--a pulse radiolysis study.

The semi-iminoquinone radical of acetaminophen, which has previously been proposed as a possible hepatotoxic intermediate in the cytochrome P-450 catalysed oxidation of acetaminophen, has been generated and studied by pulse radiolysis. In the absence of other reactive solutes, the radical decays rapidly by second order kinetics with a rate constant (2k2) of (2.2 +/- 0.4) x 10(9) M-1 sec-1. In alkaline solutions the radical deprotonates with a pK of 11.1 +/- 0.1 to form a radical-anion, as confirmed by the effect of ionic strength on the rate of radical decay. The acetaminophen radical-anion reacts with resorcinol at high pH values, leading to the formation of a transient equilibrium from which the one-electron reduction potential of the semi-iminoquinone radical of acetaminophen is estimated to be +0.707 +/- 0.01 V at pH 7. This value predicts that acetaminophen should be oxidised by thiyl radicals. This was confirmed by pulse radiolysis experiments for reaction of the cysteinyl radical, for which rate constants of 7 x 10(6) M-1 sec-1 at pH 7 and 2.7 x 10(8) M-1 sec-1 at pH 11.3 were obtained. The reaction of O2 with the acetaminophen semi-iminoquinone radical could not be detected by pulse radiolysis, and alternative mechanisms for superoxide radical formation are discussed.

Fast solute release from photosensitive liposomes: an alternative to ‘caged’ reagents for use in biological systems

The kinetics of release of soluble marker trapped in liposomes of gel phase phospholipid containing a photoisomerisable phospholipid analogue have been investigated. Marker release is triggered by UV laser flash photolysis at 355 nm. A markedly temperature‐dependent release rate is seen, and above 25°C millisecond release kinetics can be achieved. These results suggest that such liposomes might find application as an alternative to conventional ‘caged’ reagents for photo‐triggered reagent release in biological research.

ASPECTS OF CHEMICAL DAMAGE IN RADIATION AND PHOTO‐BIOLOGY

Abstract— The reactions of oxidizing free radicals produced by the action of ionizing radiation on aqueous solutions with molecules of biological interest are discussed. In particular the reactions of inorganic radical anions with proteins are considered and their use as selective probes described by example.

Photosensitive liposomes as ‘cages’ for laser‐triggered solute delivery: the effect of bilayer cholesterol on kinetics of solute release

Liposomes containing acyl chains incorporating azobenzene chromophores have been investigated as potential ‘caging’ agents for fast solute release. On photolysis, trapped marker dye can be released from gel‐phase liposomes within milliseconds. Solute release is markedly sensitive to the presence of cholesterol in the bilayer. Phospholipids bearing one saturated acyl chain and an azobenzene‐substituted chain are ineffective as sensitisers unless cholesterol is present, while doubly substituted phospholipids sensitise release in its absence. Cholesterol markedly affects the temperature profile of solute release depending on the host phospholipid chain length. Solute release is not seen for lipid hosts with unsaturated acyl chains.

Transverse distribution of α-tocopherol in bilayer membranes studied by fluorescence quenching

The fluorescence properties of alpha-tocopherol in a range of solvents and in micelles and membrane vesicles have been measured. In solvents the fluorescence decay was fitted by a single exponential. In bilayer membranes of dipalmitoylphosphatidylcholine or egg phosphatidylcholine the fluorescence decay was more accurately fitted as a double exponential. This may indicate that alpha-tocopherol occupies two or more sites in such membranes. Depth-dependent quenching of alpha-tocopherol fluorescence by acrylamide and some doxyl stearates has also been studied. The results confirm that in gel-phase lipid the chromanol group has a transverse distribution close to the head-group region of the lipid. In fluid phase lipid in the presence of buffer the results indicate there is more penetration of the chromanol group into the bilayer.

Quenching of reactive oxidative species by probucol and comparison with other antioxidants

One-electron oxidation of the antiatherogenic and antiatherosclerotic drug probucol has been studied in relation to its activity as an antioxidant. Oxidation by triplet excited states of duroquinone and benzophenone, and by the inorganic radicals Br2.- and N3., lead to the formation of a transient absorption at 500 nm. This was identified by time-resolved resonance Raman spectroscopy as the phenoxyl radical from probucol, formed by hydrogen atom or electron plus proton loss from one of the phenolic groups of probucol. The reactivity of probucol with triplet duroquinone and triplet benzophenone, and as a quencher of singlet oxygen, was compared with the reactivities of other antioxidants (alpha-tocopherol, palmitoyl ascorbic acid, dihydrolipoic acid and N-acetyl cysteine). In quenching of the triplet states the reactivity of probucol was comparable with that of alpha-tocopherol, whereas as a quencher of singlet oxygen probucol (k < 10(6) M-1 s-1) was less effective than alpha-tocopherol (k = 2.0 x 10(8) M-1 s-1) by more than two orders of magnitude. This difference in reactivity may allow the contribution of singlet oxygen towards oxidative stress to be quantified separately.

Formation of singlet oxygen from solutions of vitamin E

Vitamin E offers protection against oxidative stress and is an efficient quencher of singlet oxygen. A recent report suggests that photo-excitation of vitamin E results in the formation of a triplet state (Naqvi et al. Photochem Photobiol Sci 2, 381 (2003)). This leads to the possibility of the triplet state of vitamin E being able to sensitize singlet oxygen and if this is the case it would be counter productive in terms of the biological protective function of vitamin E. We report the production of singlet oxygen, detected by 1270 nm luminescence, from pulsed laser excitation (308 nm) of vitamin E and an analogue, 2,2,5,7,8-pentamethyl-6-hydroxy-chroman (PMHC), with quantum yields between ∼0.1 and 0.2. The luminescence was identified as singlet oxygen from self-quenching by vitamin E with solvent-dependent rate constants similar to published values. Whilst the beneficial antioxidant aspects of vitamin E are well established, these results indicate that vitamin E when directly excited can sensitize singlet oxygen formation and may, therefore, be capable of inducing biochemical and biological damage. The results are discussed in relation to recent reports on the deleterious effects of vitamin E dietary supplementation and pro-oxidant effects of vitamin E.