Haber-Weiss sixty years onPeter WardmanReview Article:Free Radicals: nature's way of saying NO or Molecularmurderfrom the 1993 Gray Laboratory Annual Reportbeing a description of the chemical basis for the generation of physiologically-active or cytotoxic species involving free radicals as molecular messengers or reactive intermediatesSummaryFree radicals are important intermediates in natural processes involved in cytotoxicity, control of vascular tone,and neurotransmission. The chemical kinetics of free-radical reactions control the importance of competingreaction pathways. Equilibria involving protons often influence the reaction kinetics of free radicals important inbiology. Radiolysis is a powerful method to generate specific free radicals and measure their reactivity. Currentwork in this area at the CRC Gray Laboratory is providing information fundamental to our understanding of themolecular biology of cytotoxic and physiological processes, and leading to the identification of new targets forexploitation of cellular chemistry.IntroductionA common misconception amongst the general public is that `natural' foods are always `good for you'. Extendingthis logic to biological processes, this raises the question as to how the body can destroy or otherwise eliminateunwanted molecules or organisms, using the normally apparently harmless molecules at its disposal. In a reviewin an earlier Annual Report I described how chemicals called free radicals derived from drugs can be designedto act as `magic bullets' in cancer chemotherapy and diagnosis, to help kill or mark specifically cells lackingoxygen in tumours (Free radicals: magic bullets in cancer therapy and diagnosis? Gray Lab. Ann. Rept., 1990,p.13; copies may be obtained from the author). This article concentrates on the chemical reactions, involvingfree radicals, the body makes use of to respond to diverse challenges, reactions which may result in unwantedinjury if the natural defences are overwhelmed.This is an area in which research has grown enormously in recent years, free-radical research now possiblyrivalling radiation research in terms of resources devoted world-wide. Chemical bonds are usually formed fromthe sharing of two electrons, whereas a free radical is a species with one unpaired electron. This makes many,but not all, free radicals chemically quite reactive, as the species seek to find another electron to pair up with.However, the definition includes common chemicals such as oxygen. Not surprisingly, therefore, oxygen is acommon reactant in free-radical processes, having a propensity to take part in single-electron transfer or free-radical addition reactions in which electrons become paired. Another common gaseous chemical which is a freeradical is nitric oxide. It is now recognized to play a critical role in vascular physiology, and with its molecularformula of NO, this has led to as many puns in reviews of its role as the diverse roles themselves. Apart from thetitle of this article, the mind soon turns to phrases such as `NO sex, please, ...' (since nitric oxide is involved inpenile erection). Biological messengers are often needed to be short-lived, degradable, controllable andreusable: the superoxide radical, the electron-adduct of superoxide, fulfils all these criteria.This article outlines some of the chemical background to the use nature makes of free radicals in ordinarybiological processes. The challenge in cancer research is to find ways of exploiting this chemistry for therapeuticgain, and some of our exploratory approaches are mentioned briefly in the report of the Molecular Mechanismsof Therapy Group. In contrast to the reductive free-radical processes stressed in the earlier Magic bullets ...review, free-radical reactions the body uses naturally are often oxidative in nature, as described below. To helpkeep the reactions in check, antioxidants to mop up unwanted free radicals have evolved, such as vitamin C(ascorbate) or vitamin E. Glutathione, a natural thiol commonly involved in coupling reactions to help eliminateunwanted chemicals by renal excretion, for example, also plays a part in the action of these antioxidants.Ionizing radiation ejects single electrons from molecules, and so the radiolysis of water, for example, generatesfree radicals at a rate readily controlled by manipulating the radiation source. By adding suitable solutes, specificfree radicals identical to those the body produces naturally can be generated. Thus techniques originallydeveloped to help study the reactive intermediates in chemical events following radiolysis have proven to bepowerful methods of characterizing the reactions of natural free radicals in predominantly aqueous media suchas the bulk of the cellular environment. In fact, the rates of several of the most important reactions of natural freeradicals were first measured in this way, and the tools of radiation chemistry provide important informationconcerning free radical reactions of biological importance. Radiation chemistry is therefore becomingincreasingly important in this much wider context.Free radicals and cellular oxidative stressOxidation and reduction are chemical terms which describe the loss or gain of electrons by molecules, respectively. Thus ferrous iron (Fe2+) is oxidized to ferric iron (Fe3+) by the loss of a single electron, the charge onthe ion changing from +2 to +3 in the process. Hydroxide ions in water (OH- ) can be ionized, losing an electron, to give hydroxyl free radicals (·OH); the unpaired electron in ·OH is denoted by the radical `dot', and such species have a strong tendency to restore the electron pair by pulling a hydrogen atom, complete with a single unpaired electron, from C-H bonds in sugars, in DNA for example. An oxidizing agent is thus a molecule, atom or radical fragment which likes to gain an electron. (In fact, radiation chemistry has provided us with a versatile and powerful method of quantifying the propensity to electron gain or loss involving short-lived free radicals, where conventional electrochemical methods fail.)In the present context, oxygen itself is one of the commonest oxidizing agents. When oxygen acts as anoxidizing agent, it gains one or more electrons from a substance. If it adds a single electron, the superoxide freeradical is formed (O2·- ). This is an extremely common substance being produced
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