Biological Free Radicals

BY GRAHAM S. TIMMINS AND MICHAEL J. DAVIES

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 1994 (when this area was last reviewed) to early 1998. It covers both direct EPR spectroscopy and spin trapping studies as well as related techniques; it does not cover recent developments in the synthesis and chemistry of spin traps, the formation and reactions of radicals in enzymes and metalloproteins, DNA damage, or spin labelling studies; these topics are covered elsewhere in this volume. Food irradiation, and its detection by EPR spectroscopy, has recently been extensively and thoroughly reviewed, and is also therefore not covered. Owing to the increasing interest in, and use of, EPR in the biomedical field, this review cannot be all encompassing and complete due to space limitations; we have, however, endeavoured to cover major advances that have occurred during this time period, and apologise for any omissions. Emphasis has been placed on novel discoveries and processes, and hence we have deliberately omitted the majority of (the very large number of) studies where EPR spin trapping has been employed in the assessment of putative antioxidant / scavenging compounds, in which (typically) the trapping of HO· or O2·- by DMPO (to give the well-characterised DMPO-OH or DMPO-OOH adducts) has been employed purely as a competitive 'clock' reaction. The very large volume of literature that has developed over the last few years on the trapping on nitric oxide (NO·), which has a wide variety of important 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 in HO· formed during the Fenton reaction (Fe2+ / H2O2) has been investigated using both H2O2 and H2O labelled with 17O and the spin trap DMPO. The DMPO-OH adduct was observed to give 17O couplings when labelled peroxide was employed, but not when the H2O contained 17O, establishing unequivocally that the oxygen atom in the trapped HO· arises solely from the peroxide. The role and selectivity of HO· in the degradation of hyaluronic acid, other polysaccharides and mono-saccharides has been examined using both direct rapid-flow and spin trapping EPR. In these studies little selectivity was observed with low-molecular-weight substrates, but only a few of the possible radicals were observed with some of the polymers; this may be due to the increased stability of some of these species and their slower rates of rearrangement. The selectivity of HO· attack on collagen, model peptides and free amino acids has also been investigated by EPR spin trapping using nitroso spin traps.

HO· generation by a series of iron-containing minerals (magnetite and haematite), an iron-exchanged zeolite, and asbestos fibres (chrysolite and crocidolite) has been investigated using DMPO as a spin trap. Zeolite and asbestos fibres proved to be effective catalysts for HO· generation, whereas the oxides were mostly inert. The total surface concentration of iron in these materials has been reported to be unrelated to the yield of HO·, as only a few of the iron ions on the surface appear to be active; this may be related to their redox state and coordination. A somewhat similar study has been carried out with coal mine dust, with all dust (from various mines in the USA) and standard samples giving rise to HO· generation in the presence of DMPO. In this case however there was a positive correlation between the rate of radical formation and surface iron content, and this extended to the ability of these dusts to induce lipid peroxidation. This catalytic activity has been suggested to be important in the development of pneumoconiosis and other pulmonary diseases in coal workers.

The ability of Fe2+/ascorbic acid mixtures to induce lipid oxidation on liposomes has been investigated using a number of different spin traps. In this system no evidence was obtained for HO· generation, with only carbon-centred radicals detected; these presumably arise from the lipid moieties. This conclusion is supported by the observation that the signal intensity of these radicals was dependent on the lipid concentration.

The role of iron ions in catalysing radical formation in vivo in chronic iron-loaded rats or cultured hepatocyte cells has been investigated. In the former study a secondary radical trapping technique was employed where the initially generated HO· reacts with added DMSO to give methyl radicals which are then trapped by PBN to give a stable radical adduct. The methyl radical adduct to PBN was detected in the bile of animals 10 weeks after being fed on an iron-loading diet and 40 minutes after i.p. injection of the spin trap. Desferal (desferrioxamine) completely inhibited this radical formation, and great care was taken to exclude artifactual radical formation ex vivo. In a second study 11 cultured hepatocytes were treated with various levels of iron and the formation of lipid-derived radicals detected using the spin trap POBN. Radical formation was found to be both time-, and iron concentration-, dependent over a 24 hour period. Two iron chelators - desferal and a hydroxypyrid-4-one (CP20) inhibited radical formation when these materials were introduced either before or simultaneously with the iron. A further chelator, pyoverdin, was not protective.

2.2 Copper – The role of two metal ion complexes - HgCl2 and [Fe(CN)6]3 - in releasing metal ions from Cu(l)-metallothioneins thereby promoting radical formation, has been investigated. Two DMPO adducts, the HO· adduct and a carbon-centred species, were detected when HgCl2 was used, but no oxygen-derived radicals were detected with [Fe(CN)6]3-. These results, in conjunction with inhibitor studies (using SOD and catalase), suggest that this mercury salt can displace copper ions from metallothioneins, and that the released ions subsequently autoxidise to give oxygen-centred...