Pharmacology and Toxicology

1. Background: Radical metabolites of foreign compounds
Free radicals play a key role in the biological actions of many xenobiotics, including outright poisons and medicines. In the case of the latter, there are examples in which radicals appear to be involved in both their therapeutic actions and their side-effects, the classical examples being the quinone antitumour agents.

Even where foreign compounds do not stimulate radical generation directly, their depletion of glutathione (e.g.during the metabolism of paracetamol) can leave the cell less able to defend itself against reactive oxygen and nitrogen species produced endogenously by normal metabolic processes. The list of chemicals whose actions appear to involve free-radical metabolites continues to grow: halogenated hydrocarbons (e.g. halothane and carbon tetrachloride), alcohols (ethanol and methanol), bipyridyl herbicides (paraquat and diquat), tobacco-smoke components, alloxan, 6-hydroxydopamine, nitro compounds, benzene, phenol and numerous quinones and hydroquinones, to name but a few.

Many quinones and nitro compounds participate in redox-cyling reactions ('futile cycling') involving reductase enzymes: typically a semiquinone or nitro radical anion is formed, which is then oxidised by molecular oxygen, producing the superoxide anion radical (O2). The parent quinone or nitro compound, re-generated in the reaction with oxygen, is then reduced back to the corresponding radical (typically by NADPH-cytochrome P450 reductase), only for the whole process to be repeated. This results in a continuous flux of superoxide radical generation.

Nitrofurantoin, an anti-bacterial drug, is an good example of a futile-cycling agent. Bacterial reductases reduce the drug to a nitro radical anion, which then donates its extra electron to oxygen, producing the superoxide radical. However similar reactions (involving reduction by NADPH-cytochrome P450 reducase) are believed to be responsible for the pulmonary toxicity of the drug. In both situations, metal ions can promote the conversion of superoxide to the hydroxyl radical (OH), which is able to attack DNA and other cellular targets (see 'Metal Ions in Health and Disease' ):

2. Expertise and services offered by Westcott Research and Consulting
Dr Burkitt has some 20 years' laboratory experience investigating the involvement of free radicals in the biological actions of foreign compounds. This included post-doctoral training in the Laboratory of Molecular Biophysics Free-Radical Metabolite Workgroup at the NIH (sponsored by Prof. R. P. Mason) and in the Division of Toxicology at the Karlolinska Institute (Prof. S. Orrenius). More recent experience includes that obtained in the Cancer Research UK Free Radicals in Cancer Research Group (1999 2006).

In addition to a number of original research articles in this area, Dr Burkitt has written two Specialist Periodical Reports for the Royal Society of Chemistry on free-radical metabolites in biology and medicine (details below). He has also refereed numerous articles and grant applications in this field.

Westcott Research and Consulting offers advice and expertise in all aspects of the free-radical metabolism of foreign compounds, including:

In summary, Dr Burkitt can assist in establishing free-radical mechanisms of toxicity that involve the linking of fundamental chemical reaction mechanisms with biological outcomes. Below are described two such examples of the many projects to which Dr Burkitt has contributed.

2.1 Mechanism of action of dithiocarbamate compounds
The dithiocarbamates, such as N,N-diethyldithiocarbamate (DDC) and pyrrolidine dithiocarbamate (PDTC), are a group of sulphur-containing metal-chelating agents with radical-scavenging properties. They have found widespread application in agriculture where are they are used as insecticides, fungicides and herbicides and in medicine as antidotes to metal poisoning and as anti-microbial agents. They have also been used in the experimental treatment of HIV infection. PDTC, in particular, is also employed widely in molecular and cellular biology, where it is known to inhibit apoptosis and the transcription factor NF-κB.

Due to their possession of a sulphydryl group (R-SH), dithiocarbamates are powerful radical-scavenging antioxidants (see 'Antioxidant Chemistry' ). This property was widely assumed to underlie their effects on apoptosis and NF-κB activation, which helped embed the view that reactive oxygen and nitrogen species serve as cell-signalling agents in these processes. However, the discovery by Professor S. Orrenius and colleagues at the Karolinska Institute that the incubation of thymocytes with PDTC can result in the induction of oxidative stress involving the uptake of copper ions from the growth medium and the subsequent oxidation of glutathione to its disulphide (for details of this process, see 'Antioxidant Chemistry' ), leading to the initiation of apoptosis necessitated a re-evaluation of the mechanisms by which dithiocarbamates affect cells.

Working in collaboration with Professor Orrenius' team, Dr Burkitt and his collegues helped elucidate the chemical reactions underlying the biological actions of dithiocarbamates. It was shown that these compounds form complexes with Cu(II) ions present in growth media. The metal ion is then reduced and transported into cells. E.g. for DDC:

2 (C2H5)2NCS(S)SH + Cu2+ → (C2H5)2NC(S)S-S(S)CN(C2H5)2 + Cu+

Although the Cu(I) ions formed in this reaction are able to undergo limited redox cycling, this is not associated with the release of reactive oxygen species, which are scavenged in situ by the excess thiol (for further details of these reactions, see 'Metal ions in Health and Disease' and 'Antioxidant Chemistry' ).

However during their reduction of Cu(II) to Cu(I), dithiocarbamates are oxidised to their corresponding thiuram disulphides (see equation above). These disulphides ('disufiram' in the case of DDC) are responsible for many of the biological actions of dithiocarbamates. In short-term incubations, thiuram disulphides can inhibit apoptosis by forming mixed disulphides with caspase-3 (believed to involve the catalytically-active cysteinyl-163 residue) and/or upstream processors responsible for cleaving the pro-enzyme. Upon prolonged incubation, however, thiuram disulphides can induce cell death, which involves their oxidation of glutathione, as shown below for disulfiram (and its parent dithiocarbamate, DCC):

2.2 An evaluation of the safety of hydroxyurea in the treatment of myeloproliferative disorders
A range of myelosuppressive agents, including hydroxyurea, busulfan, chlorambucil and radiophosphorus (32P), are employed to reduce the risk of thrombosis in patients with polycythaemia vera and essential thrombocythaemia. Although many such agents are known to increase the risk of transformation to acute myeloid leukaemia, the leukaemogenic potential of hydroxyurea has been the subject of much debate. Studies using cells grown in culture have shown that hydroxyurea can induce DNA damage, suggesting the agent certainly has the potential to bring about transformation.

In response to an enquiry, Dr Burkitt and a consultant clinical haematologist colleague (A. Raafat) made an assessment of the risk of using hydroxyurea in the management of myeloproliferative disorders. Rather than examine anecdotal patient records and the various statistical analyses on the risk of acute myeloid leukaemia in individuals receiving the drug, they looked for clues in the chemistry underlying the biological actions of hydroxyurea.

Hydroxyurea suppresses cell proliferation by inhibiting the enzyme ribonucleotide reductase, thereby limiting the supply of deoxyribonucleotide 5'-triphosphates for DNA synthesis. Although hydroxyurea can inhibit the reductase directly, there is evidence of a role for the nitric oxide radical (NO). Nitric oxide is generated via the three-electron oxidation of hydroxyurea, which is expected to be brought about by myeloperoxidase and prostaglandin H synthase (cyclooxygenase) in the bone marrow:

The nitric oxide radical is believed to inactivate ribonucleotide reductase by reacting with a protein tyrosyl radical involved in the reaction cycle of the enzyme. However nitric oxide can also react with the superoxide radical, resulting in the formation of peroxynitrite (ONOO), which can be converted to a variety of 'reactive nitrogen species' including the nitrogen dioxide radical (NO2) capable of inflicting damage to the cell, including DNA damage.

Under normal physiological (non-inflammatory) conditions, the concentration of superoxide in cells may not be high enough to react with nitric oxide generated from hydroxyurea. However nitrogen dioxide, which is believed to be responsible for much of the damage sustained by cells exposed to nitric oxide under pathological conditions, can also be generated by reaction of the latter with molecular oxygen:

2 NO + O2 → 2 NO2

As set out in the 2006 Blood article listed below, it was suggested that the DNA damage seen in cultured cells exposed to hydroyxurea results from the conversion of NO to the highly reactive NO2 radical upon reaction with oxygen. Moreover, it was argued that, due to the much lower concentrations of oxygen in vivo, such reactions would not occur in patients receiving hydroxyurea in cytoreductive therapy. In other words, it was proposed that generation of the mutagen NO2 is peculiar to cell culture systems, where the cells are typically grown under abnormally high, non-physiological oxygen partial pressures.

On this basis (argued from chemical kinetics) it was concluded that hydroxyurea is unlikely to be mutagenic in vivo when used in the management of myeloproliferative disorders.

Selected publications showing the scope of Dr Burkitt's experience in this area: