1.1 Background: Historical aspects of cell killing by radicals
Prior to the discovery of the Cu,Zn-superoxide dismutases by Irwin Fridovich and Joe McCord in the late 1960s (see 'Spectroscopy' ) it was generally believed that damage to cells and their components involving free radicals was confined to the field of radiation biology, where it is recognised that ionising radiation (X-rays and γ-radiation) can bring about the generation of radicals by two general mechanisms: (i) the direct ionisation of molecules, such as DNA and proteins; and (ii) the radiolytic cleavage ('radiolysis') of water molecules, which results in the generation of a variety of reactive species including the hydrated electron (e–(aq)), the hydroxyl radical (•OH), the hydrogen atom (•H) and hydrogen peroxide (H2O2).
The killing of cells exposed to ionising radiation was believed to occur as a direct result of the damage resulting from radical generation on biological molecules (including DNA, proteins and lipids), either through direct ionisation or attack by the products of water radiolysis. It was quite natural, then, that in the years following McCord and Fridovich's landmark discovery researchers looked to the field of radiation biology for clues as to how oxygen radicals generated by endogenous processes - without the intervention of ionising radiation - might bring about cell damage and killing. Interest in the mechanisms through which such species kill cells only intensified as the list of foreign compounds - including many pharmacological agents - demonstrated to promote radical generation in vivo lengthened by the year (see also 'Pharmacology and Toxicology').
1.2 The importance of reactive oxygen species in apoptosis
Whilst there is no doubt that at high concentrations oxygen radicals - and various related, non-radical 'reactive oxygen species', such as hydrogen peroxide - can bring about cell death via their direct, destructive effects on key biological molecules and structures (including DNA and cell membranes), it is largely through the work of Sten Orrenius and his colleagues at the Karolinska Institute that we owe our recognition of the importance of apoptosis as a mechanism for the killing of cells exposed to such species.
The term apoptosis was introduced by Andrew Wyllie and colleagues in 1972 to describe a mode of cell death, defined at the time by morphological criteria, that is controlled and driven largely by endogenous processes: essentially, the cell plays an active role in bringing about its own demise. Apoptosis occurs widely in biology, being involved in a diverse range of phenomena, from the selection of T-cells in the thymus ('thymic education') to the shedding of leaves by deciduous trees. The process can also occur in stressed or damaged cells, which has led to it being referred to as 'altruistic suicide' (e.g. the self-deletion of a precancerous cell by apoptosis would be in the interests of the organism as a whole, if not the individual cell).
Professor Orrenius recognised the similarities between cells undergoing apoptosis, as described by Wyllie, and those exposed to reactive oxygen species. The efforts of Orrenius and numerous other researchers since have led to our current awareness and understanding of the important role played by reactive oxygen species in apoptosis. Not only are they able to induce the biomolecular damage which can trigger cells to undergo apoptosis, these species serve as cell-signalling agents in many of the key regulatory pathways that act to control cell survival, proliferation and indeed deletion. Examples include the pathways involving protein kinase C, apoptosis-regulating kinase 1 (ASK-1), Bcl-2 and the mitogen-activated protein kinases (MAPKs), to name but a few.
Since the 1970s, then, we have gone through a whole circle from viewing oxygen radicals and other reactive oxygen intermediates as wholly harmful species, generated by poisons or 'accidentally' by endogenous processes, to their recognition as cell-signalling species that play crucial roles in many important cellular processes.
2. Expertise and services offered by Westcott Research and Consulting
Dr Burkitt undertook post-doctoral training in the laboratory of Professor Sten Orrenius at the Karolinska Institute. He has since collaborated with Professor Orrenius on projects requiring expertise in the chemical aspects of free radical metabolism, a good example being the elucidation of the mechanisms through which dithiocarbamate compounds modulate cell death (see 'Pharmacology and Toxicology').
Other areas in which Dr Burkitt has acquired experience and offers expertise through Westcott Research and Consulting include the following:
In the experiment shown below, the activity of recombinant caspase-3 was monitored by its ability to cleave an artificial substrate, releasing the fluorescent molecule AFC. The traces show how hydrogen peroxide can inactivate the enzyme. This can be fully reversed using DTT, which regenerates the reduced thiol group (not shown). The top traces show how - after the removal of excess H2O2 using glutathione peroxidase, GtPx - the caspase can be partially re-activated by thioredoxin and its reductase (Trx + TR). The failure of Trx/TR to re-activate the enzyme in the presence of cytochrome c, which is released from mitochondria during apoptosis, is believed to reflect the generation of a peroxidase compound I-type intermediate, which brings about the irreversible, single-electron oxidation of the protein thiol to a thiyl radical (Cys-S•). This radical becomes 'fixed' by the addition of oxygen (forming Cys-S-OO•), rendering the damage irreversible.
Selected publications showing the scope of Dr Burkitt's experience in this area: