The characterisation and quantification of O6-alkylguanine adducts in DNA

In the modern world, exposure to artificial chemicals is inevitable through food and drink, air pollution, and consumer products. Some of these chemicals can damage DNA, increasing the risk of disease. Exposure to alkylating agents, such as N-nitroso compounds, leads to the formation of mutagenic and carcinogenic O6-alkylguanine adducts (O6-AlkGs), which are implicated in the development of colorectal cancer (CRC).
In this work, O6-methylguanine and O6-carboxymethylguanine adducts were characterised and quantified in human CRC tumour DNA using stable isotope dilution mass spectrometry (SID-MS). To our knowledge, this is the first study to apply SID-MS to compare O6-AlkG levels in CRC tumour DNA with matched healthy DNA from the same patients. Naturally occurring 14N and isotopically labelled 15N nucleoside standards of these adducts, along with O6-ethyl, carboxyethyl, benzyl, and pyridyloxobutyl guanine adducts, were synthesised. Selected adducts were incorporated into oligodeoxyribonucleotides to develop digestion and quantification methodologies for SID-MS analysis. Although all synthesised adducts were intended for analysis in human DNA, progress was limited by time constraints imposed by the COVID-19 pandemic.
Bracken fern (Pteridium aquilinum), the most widespread fern globally, is the only known carcinogenic plant. Widespread grazing exposes humans to the carcinogen ptaquiloside (PTQ) through red meat consumption, dairy product consumption and groundwater contamination. We hypothesised that PTQ’s carcinogenicity arises from the formation of O6-alkylguanine adducts, specifically O6-pterosin B guanine (O6-PTBG). Reaction of the active PTQ dienone with duplex DNA followed by digestion revealed the presence of O6-PTBG by LC-MS. The corresponding nucleoside standard was synthesised, and an O6-PTBG-containing oligodeoxyribonucleotide was prepared to develop digestion methodology. This study represents the first detection of the O6-PTBG adduct formed from the reaction of PTQ dienone with duplex DNA.