Steven A. Everett

Comparative mechanisms and rates of free radical scavenging by carotenoid antioxidants

The comparative mechanisms and relative rates of nitrogen dioxide (NO2 ⋅), thiyl (RS⋅) and sulphonyl (RSO2 ⋅) radical scavenging by the carotenoid antioxidants lycopene, lutein, zeaxanthin, astaxanthin and canthaxanthin have been determined by pulse radiolysis. All the carotenoids under study react with the NO2 ⋅ radical via electron transfer to generate the carotenoid radical cation (Car⋅+). In marked contrast the glutathione and 2‐mercaptoethanol thiyl radicals react via a radical addition process to generate carotenoid‐thiyl radical adducts [RS‐Car]⋅. The RSO2 ⋅ radical undergoes both radical addition, [RSO2‐Car]⋅ and electron abstraction, Car⋅+. Both carotenoid adduct radicals and radical cations decay bimolecularly. Absolute rate constants for radical scavenging were in the order of ∼107–109 M−1 s−1 and follow the sequence HO(CH2)2S⋅>RSO2 ⋅>GS⋅>NO2 ⋅. Although there were some discernible trends in carotenoid reactivity for individual radicals, rate constants varied by no greater than a factor of 2.5. The mechanism and rate of scavenging is strongly dependent on the nature of the oxidising radical species but much less dependent on the carotenoid structure.

Oxidation of tetrahydrobiopterin by biological radicals and scavenging of the trihydrobiopterin radical by ascorbate.

One-electron oxidation of (6R)-5,6,7,8-tetrahydrobiopterin (H(4)B) by the azide radical generates the radical cation (H(4)B(*)(+)) which rapidly deprotonates at physiological pH to give the neutral trihydrobiopterin radical (H(3)B(*)); pK(a) (H(4)B(*)(+) <==> H(3)B(*) + H(+)) = (5.2 +/- 0.1). In the absence of ascorbate both the H(4)B(*)(+) and H(3)B(*) radicals undergo disproportionation to form quinonoid dihydrobiopterin (qH(2)B) and the parent H(4)B with rate constants k(H(4)B(*)(+) + H(4)B(*)(+)) = 6.5 x 10(3) M(-1) s(-1) and k(H(3)B(*) + H(3)B(*)) = 9.3 x 10(4) M(-1) s(-1), respectively. The H(3)B(*) radical is scavenged by ascorbate (AscH(-)) with an estimated rate constant of k(H(3)B(*) + AscH(-)) similar 1.7 x 10(5) M(-1) s(-1). At physiological pH the pterin rapidly scavenges a range of biological oxidants often associated with cellular oxidative stress and nitric oxide synthase (NOS) dysfunction including hydroxyl ((*)OH), nitrogen dioxide (NO(2)(*)), glutathione thiyl (GS(*)), and carbonate (CO(3)(*-)) radicals. Without exception these radicals react appreciably faster with H(4)B than with AscH(-) with k(*OH + H(4)B) = 8.8 x 10(9) M(-1) s(-1), k(NO(2)(*) + H(4)B) = 9.4 x 10(8) M(-1) s(-1), k(CO(3)(*-) + H(4)B) = 4.6 x 10(9) M(-1) s(-1), and k(GS(*) + H(4)B) = 1.1 x 10(9) M(-1) s(-1), respectively. The glutathione disulfide radical anion (GSSG(*-)) rapidly reduces the pterin to the tetrahydrobiopterin radical anion (H(4)B(*-)) with a rate constant of k(GSSG(*-) + H(4)B) similar 4.5 x 10(8) M(-1) s(-1). The results are discussed in the context of the general antioxidant properties of the pterin and the redox role played by H(4)B in NOS catalysis.

Nitric oxide in biological fluids: analysis of nitrite and nitrate by high-performance ion chromatography

The analysis of nitric oxide-derived nitrite and nitrate ions in biological fluids represents a proven strategy for determining nitric oxide participation in a diverse range of physiological and pathophysiological processes in vivo. In this article we describe a versatile method for the simultaneous measurement of NO2- and NO3- anions in both plasma and isolated tumour models based on anion-exchange chromatography with spectrophotometric detection (214 nm). This method compares well with the capillary electrophoresis technique, exhibiting an equivalent sensitivity for NO2-/NO3- anions and short run-times, i.e. not greater than 4 min. Comparisons are also made with two alternative but less satisfactory methods which employ ion-exchange or reversed-phase ion-pair chromatography with conductimetric as well as spectrophotometric detection. Technical problems associated with each method, particularly those arising from nitrate contamination, have been addressed.

FREE-RADICAL REPAIR BY A NOVEL PERTHIOL: REVERSIBLE HYDROGEN TRANSFER AND PERTHIYL RADICAL FORMATION

2-(3-Aminopropyl-amino) ethaneperthiol (RSSH, the perthiol analogue of the thiol radioprotector, WR-1065) reacts with the alpha-hydroxy alkyl radical (CH3)2C.OH by donating a hydrogen atom as indicated by the characterization of perthiyl radicals (RSS.; lambda max approximately 374 nm, epsilon 374 approximately 1680 +/- 20 dm3 mol-1 cm-1) by pulse radiolysis. The perthiyl radical abstracts a hydrogen from the alcohol to establish a reversible hydrogen-transfer equilibrium. This equilibrium lies predominantly on the side of radical repair since the rate constants for the forward and reverse reactions at pH 4 are: kappa(RSSH+(CH3)2C.OH) = (2.4 +/- 0.1) x 10(9) dm3 mol-1 s-1 and kappa(RSS.+(CH3)2CHOH) = (3.8 +/- 0.3) x 10(3) dm3 mol-1 s-1 respectively. The pKa (RSSH<-->RSS(-)+H+) = 6.2 +/- 0.1 was determined from the pH dependence of the rate of perthiol repair. Identical experiments have been performed with WR-1065 allowing a direct comparison of free-radical repair reactivity to be made with the parthiol analogue. At pH approximately 7.4 the reactivities of the thiol and perthiol were similar, both repairing the alcohol radical with a rate constant of approximately (2.4 +/- 0.1) x 10(8) dm3 mol-1 s-1. However, at pH 5 whilst the hydrogen-donation rate of the thiol was 15-20% higher than at pH 7.4, the perthiol reactivity was over an order of magnitude higher. The thermodynamic driving force for the observed enhanced free-radical repair reactivity of RSSH compared to RSH is attributed to the resonance stabilization energy of 8.8 kJ mol-1 within the RSS. radical. These results indicate a possible application of RSSH/RSS- as DNA-targeted antioxidants or chemoprotectors.

Molecular modelling of human CYP1B1 substrate interactions and investigation of allelic variant effects on metabolism.

Molecular modelling of human CYP1B1 based on homology with the mammalian P450, CYP2C5, of known three-dimensional structure is reported. The enzyme model has been used to investigate the likely mode of binding for selected CYP1B1 substrates, particularly with regard to the possible effects of allelic variants of CYP1B1 on metabolism. In general, it appears that the CYP1B1 model is consistent with known substrate selectivity for the enzyme, and the sites of metabolism can be rationalized in terms of specific contacts with key amino acid residues within the CYP1B1 heme locus. Furthermore, a mode of binding interaction for the inhibitor, alpha-naphthoflavone, is presented which accords with currently available information. The current paper shows that a combination of molecular modelling and experimental determinations on the substrate metabolism for CYP1B1 allelic variants can aid in the understanding of structure-function relationships within P450 enzymes.

Pitfalls in the Use of Common Luminescent Probes for Oxidative and Nitrosative Stress

Lucigenin (LC2+, bis-N-methylacridinium) and 2′,7′-dichlorofluorescin (DCFH2) are widely used as chemiluminescent or fluorescent probes for cellular oxidative stress, to reflect levels of superoxide (O2·−) and hydrogen peroxide, respectively. We report mechanistic studies that add to the growing evidence for the unsuitability of either probe except in very well-defined circumstances. The ability for lucigenin to generate superoxide via reduction of LC2+ to LC·+ and redox cycling with oxygen depends on the reduction potential of the LC2+/LC·+ couple. Redox equilibrium between LC·+ and the redox indicator benzyl viologen is established in microseconds after generation of the radicals by pulse radiolysis and indicated E(LC2+/LC·+) ∼ −0.28 V vs. NHE. Reaction of LC·+ with O2 to generate O2·− was also observed directly similarly, occurring in milliseconds, with a rate constant k ∼ 3 × 106M−1 s−1. Quinones act as redox mediators in LC·+/O2 redox cycling. Oxidation of DCFH2 to fluorescent DCF is not achieved by O2·− or H2O2, but NO2·) reacts rapidly: k ∼ 1 × 107M−1 s−1. Oxidation by H2O2 requires a catalyst: cytochrome c (released into the cytosol in apoptosis) is very effective (even 10 nM). Fluorescence reflects catalyst level as much as O2·−) production.

The role of nitric oxide in cancer. Improved methods for measurement of nitrite and nitrate by high-performance ion chromatography

The short lifetime of nitric oxide (NO) in vivo impedes its quantitation directly; however, the determination of nitrite and nitrate ions as the end-products of NO oxidation has proven a more practical approach. High-performance ion chromatographic analysis of nitrite in biological fluids is hampered by the large amount of chloride ion (up to approximately 100 mmol/l) which results in insufficient peak resolution when utilizing conductimetric detection. Analysis of both anions in small sample volumes is also constrained by the need to minimise sample handling to avoid contamination by environmental nitrate. We report a means to remove Cl- ions from small sample volumes using Ag+ resin which facilitates quantitation of either nitrite and nitrate anions in biological samples, using silica or polymer based ion-exchange resins with conductimetric or electrochemical and spectrophotometric detection. Including a reversed-phase guard column before the anion-exchange guard and analytical column also greatly extends column lifetime.

[5] Perthiols as antioxidants: Radical-scavenging andprooxidative mechanisms

Publisher Summary Thiols (RSH) are recognized for their radical-scavenging role in protection against cellular oxidative stress and in the repair of radical-induced DNA damage. The administration of exogenous thiol drugs offers defense against many free radical–associated diseases and protects normal tissues in cancer therapy using radiation or drugs. The antioxidant efficiency of perthiols reflects the free radical-scavenging ability of perthiols and prooxidative effects associated with perthiyl radical (RSS) formation. The methods used for investigating thiol free radical reactivity can be equally applied to assess the radical-scavenging and prooxidative properties of perthiols. Pulse radiolysis has proved to be a useful tool to quantify the free radical–scavenging ability of perthiols. Chemical models have been designed to probe the interaction of perthiols with free radical species commonly associated with cellular oxidative stress and to elucidate subsequent prooxidative reactions. The free radical–scavenging reactions of perthiols are qualitatively similar to, but quantitatively different from, those of the corresponding thiol antioxidants. Perthiols are not only more efficient hydrogen donors than thiols, but as perthiolate anions they are highly efficient electron donors. The antioxidant-derived radical, in this case the perthiyl radicals, are significantly less reactive than their thiyl radical counterparts and are less likely to pose a threat if generated within the cellular environment. In view of the essential role thiols play in controlling cellular oxidative stress, the encouraging performance of perthiols as free radical scavengers suggests they will be of use as exogenous antioxidants.

Bioreductively-activated prodrugs for targeting hypoxic tissues: Elimination of aspirin from 2-nitroimidazole derivatives

2-Nitroimidazoles were synthesised substituted with aspirin or salicylic acid, as leaving groups linked through the (imidazol-5-yl)methyl position. Activation of aqueous solutions by CO2*- (a model one-electron reductant) resulted in release of aspirin or salicylate, probably via the 2-hydroxyaminoimidazole. The analogous 2-nitroimidazole with bromide as leaving group eliminated bromide in < 1 ms via the radical-anion.

Synthesis and biological properties of bioreductively targeted nitrothienyl prodrugs of combretastatin A-4

Nitrothienylprop-2-yl ether formation on the 3′-phenolic position of combretastatin A-4 (1) abolishes the cytotoxicity and tubulin polymerization-inhibitory effects of the drug. 5-Nitrothiophene derivatives of 1 were synthesized following model kinetic studies with analogous coumarin derivatives, and of these, compound 13 represents a promising new lead in bioreductively targeted cytotoxic anticancer therapies. In this compound, optimized gem-dimethyl α-carbon substitution enhances both the aerobic metabolic stability and the efficiency of hypoxia-mediated drug release. Only the gem-substituted derivative 13 released 1 under anoxia in either in vitro whole-cell experiments or supersomal suspensions. The rate of release of 1 from the radical anions of these prodrugs is enhanced by greater methyl substitution on the α-carbon. Cellular and supersomal studies showed that this α-substitution pattern controls the useful range of oxygen concentrations over which 1 can be effectively released by the prodrug. [Mol Cancer Ther 2006;5(11):2886–94]