Biological Free Radicals

BY MICHAEL J. DAVIES AND GRAHAM S. TIMMINS

1 Introduction and Scope of Review

This review covers recent literature on the use of EPR techniques to investigate the formation and reactions of radicals in biochemical, biological and medical systems during the period 1998 (when this area was last reviewed) to early 2000. It covers both direct EPR spectroscopy and spin trapping studies as well as related techniques; it does not cover metalloprotein systems, DNA damage or spin labelling studies; these topics are covered elsewhere in this volume and the previous volume. Owing to the increasing interest in, and use of, EPR in the biomedical field, this review cannot be all encompassing because of space limitations; we have however endeavoured to cover all the major advances that have occurred during this period, and apologise for any omissions. Emphasis has been placed on novel reactions, and we only cover briefly the large number of studies where spin trapping has been employed in the assessment of putative antioxidant/scavenging compounds, in which the trapping of HO· or O2·- by DMPO* (to give the DMPO-OH or DMPO-OOH adducts) has been employed solely as a competitive 'clock' reaction. The large volume of literature that has developed over the last few years on the trapping of nitric oxide (NO·), which has a wide variety of biological functions, is reviewed briefly, with particular emphasis on EPR methods. The literature covered in this review has been subdivided in a manner similar to that in previous reviews of this area with the sub-sectioning dictated by the exogenous/endogenous compounds or stimuli which result in radical formation, rather than the identity of the radicals so formed.

2 Metal Ions

2.1 Iron. – The origin of the oxygen atom present in HO· generated by the Fenton reaction has been shown by use of 17O labelling and spin trapping with DMPO, to come from the starting H2O2 rather than from water molecules, and that there is no exchange between these species. The intermediacy of ferryl species in tbese reactions could not however be eliminated if the ferryl oxygen atom arose from the labelled H2O2. Other workers have carried out a kinetic analysis of the Fenton reaction by use of DMPO spin trapping, and concluded that the time-dependent loss of DMPO-OH is due to reaction with Fe3+ and not Fe2+. The effect of chelation by oxalate on the rate of reaction of Fe2+ with H2O2 and O2 has been examined by rapid-flow EPR with t-BuOH as a radical scavenger. Using this method, rate constants of 1 × 104 M-1 s-1 and 3.6 M-1 s-1 have been determined for these two processes respectively. Reaction with O2 has been shown to be highly pH dependent consistent with the reactive species being [Fe2+(oxalate)2]2-. Reaction of this complex with H2O2 has also been shown to give rise to CO2-. by use of DMPO as a spin trap. Studies on the significance of reaction of iron complexes with O2versus H2O2 have concluded that when the ratio of O2/H2O2 is >100 then the reaction of Fe2+ with O2 becomes an important route to the formation of detrimental radicals in biological systems. Other studies have examined the effect of a range of agents on the extent of HO· formation by iron-containing systems using spin trapping, primarily with DMPO, as the means of assessing radical yields. Thus α-hydroxyacids were found to significantly enhance HO· formation, whereas other hydroxyacids, ketoacids, organic acids and aldehydes did not; the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid was found to diminish oxidant formation,8 and Lu-Duo-Wei, an extract from Chinese green tea, decreased HO· formation; the effect of this extract was greater than with tea polyphenols alone, suggesting that other compounds also played a role. In contrast, furanone compounds present in soy sauce have been shown to increase DNA damage and enhance HO· formation.

The effect of a series of novel iron chelating agents developed for the potential treatment of β-thalassemia major on HO· formation from the Fenton reaction has been investigated using DMPO; these pyridoxal- and salicylalde-hyde-isonicotinoyl hydrazone compounds decrease radical formation and enhance auto-oxidation of the Fe2+ complex to the Fe3+ form, consistent with these chelators altering the redox potential of the iron couple, but not the radicals formed. The use of spin trapping reactions, with DMPO and PBN as traps for HO· and 1-hydroxyethyl radicals respectively, but with GC/MS as the detection method for the adduct species, has been investigated. It has been reported that after suitable extraction and derivatisation, these adducts can be readily quantified and the technique provides a sensitive assay method for the generation of these species. The use of ethanol with the detection of 1- hydroxyethyl radicals by spin trapping with POBN as a method of examining HO· formation in complex biological systems has continued to be employed. Thus it has been reported that E. coli lacking SOD are not only more susceptible to DNA damage and cell killing by H2O2, but also contain higher levels of intracellular redox-active iron, as judged by enhanced levels of the POBN adducts of 1-hydroxyethyl radicals, generated as a result of HO· formation via Fenton chemistry. Pre-incubation of the bacteria with the iron-chelator desferrioxamine reduced the concentration of spin adducts detected. This technique has also been employed to examine HO· formation in chondrocyte cells stimulated with phorbol esters in the presence of Fe2+ and ethanol; the formation of the observed radicals required the presence of iron, suggesting that a Fenton-type reaction is responsible for radical formation. Similar radical formation was observed with un-stimulated fresh human and rabbit cartilage pieces in the presence of Fe2+; this may be due to metal auto- oxidation. The chelator and dye Quin2 was found to have variable effects depending on the assay system used, with this compound enhancing oxidant formation in a Fe3+/H2O2 system when examined using this POBN/ethanol system as a trap for oxidants.

The mutagenicity of p-aminophenol in E coli has been ascribed to the formation of HO· (as detected by DMPO spin trapping) as a result of interaction of the phenol with Fe3+. The formation of this radical adduct was inhibited by HO·...