Synthesis and characterisation of DNA adducts derived from the bracken toxin ptaquiloside.

Bracken is a highly invasive plant found extensively in the UK and Europe with a well-documented toxicity in cattle and other animals. Bracken toxicity derives from the presence of ptaquiloside, a norsesquiterpenoid glucoside that is both mutagenic and carcinogenic. Ptaquiloside can be transmitted to humans following direct exposure from contaminated surface or ground water in bracken infested areas, or indirectly for example from contaminated dairy products. At slightly alkaline pH ptaquiloside decomposes to form a potent alkylating agent known as bracken dienone. The exposure of DNA to bracken dienone produces N3-adenine and N7-guanine base adducts. In contrast, the corresponding O6-alkylguanine adducts (O6-(pta)G adducts) have not been characterized in DNA, although the biological consequences of their formation are substantially more significant: O6-alkylguanine adducts are promutagenic and carcinogenic, typically coding for the misincorporation of thymine during DNA replication leading to GC to AT transition mutations.

This proposal seeks the first characterization of O6-alkylguanine (specifically O6-(pta)guanine) adducts in DNA following exposure to ptaquiloside/bracken dienone that we will extract and purify from bracken. DNA containing O6-(pta)guanine adducts will be characterised using the O6-alkylguanine-binding protein Atl1 and by comparison with a synthesized O6-modified deoxyribonucleoside standard following enzymatic digestion of O6-(pta)guanine-containing DNA. Integral to this proposal, we will develop chemical syntheses of the naturally occurring enantiomer of pterosin B and novel ODNs containing O6-(pta)guanine adducts. The ability of the protein O6-methylguanine-DNA methyltransferase (MGMT) to repair O6-(pta)guanine-containing DNA and the substrate properties of this adduct for human DNA polymerase enzymes will be examined. The reaction of ptaquiloside or bracken dienone with water (decomposition) or the bases of DNA produces pterosin B and derivatives thereof. We aim to develop methods for the detection of such pterosin B-containing compounds. Our proposal will lead to a much more coherent understanding of the nature and risk to humans of exposure to bracken and its constituents.

The immediate benefit of this proposal will be to the academic community in the UK and internationally in the research areas related to the proposal: Organic and nucleic acid chemistry, DNA damage and repair. See academic beneficiaries section for more detail.

The toxicity of ptaquiloside is well documented and has direct consequences for both the environment and food quality. Our work will provide important information about the role of ptaquiloside in the formation of promutagenic adducts in DNA, the consequences of such DNA damage and will begin to evaluate a new sensitive assay for quantifying the products derived from ptaquiloside, including such DNA damage. A number of leading research groups are currently working with environment agencies in the UK, Denmark, Spain and Italy to assess the consequences of bracken toxicity and its impact on human health. We expect that our work will be of direct interest to agricultural (milk and meat) and environmental agencies (water contamination), but we are also aware of a number of SMEs interested in developing assays to test for ptaquiloside and products thereof in water, plasma, milk or indeed human tissue. We have established good links to Pharmaceutical, Biotechnology and Healthcare companies in the diagnostic area that would provide valuable contact points for potential commercialisation of methods or materials that we produce. These interactions together with the outputs from our research will therefore provide better information about the risk of ptaquiloside exposure to humans, therefore improving health and the quality of life for UK citizens, although this impact may not be realized until after the completion of the project.

The researcher employed during the project will gain further experience in synthetic chemistry but the multidisciplinary nature of the project will allow them to gain skills in nucleic acid chemistry (DNA synthesis and purification), biological chemistry (natural product extraction and purification) and molecular biology (chemical modification and analysis of DNA, protein recognition and repair of O6-alkylguanine adducts and in the use of specific immunoassay detection methods). Our research group works closely with other members of the Chemical Biology research cluster in our department, is a member of the Krebs Institute and the newly established Sheffield Centre for Nucleic Acid and Genome Biology thereby exposing the researcher to a wide range of other research. In addition both will have access to all staff training and development courses in Sheffield including our research leaders programme.