Immunoslot blot assays have been used for the analysis of many DNA adducts but problems are frequently encountered in achieving reproducible results. Each step of the assay has been systematically examined and it was found that the major problems are in the DNA fragmentation step and the use of the manifold apparatus. Optimisation was performed upon both the malondialdehyde-deoxyguanosine adduct (M 1 dG) and the O 6 -carboxymethyldeoxyguanosine adduct (O 6 CMdG) to demonstrate the applicability to other DNA adducts.Blood samples from the EPIC study (n = 162) were analysed for M 1 dG adducts and the data showed no correlation with adduct levels in other tissues indicating that the EPIC blood samples were not useful for studying M 1 dG adducts. Blood samples from a processed meat vs vegetarian diet intervention (n = 6) were analysed for O 6 CMdG and many were below the limit of detection. The reduction of background adduct levels in standard DNA was investigated using chemical and whole-genome amplification approaches. The latter gave a sensitivity improvement of 2.6 adducts per 10 7 nucleotides for the analysis of O 6 CMdG.Subsequent reanalysis for O 6 CMdG showed a weakly significant increase in O 6 CMdG on the processed meat diet compared with the vegetarian diet, demonstrating that further studies are warranted.

Abstract:The role of n-6 polyunsaturated fats upon the formation of the mutagenic DNA adduct malondialdehydedeoxyguanosine (M 1 dG) in blood was investigated in male volunteers (n = 13) who consumed diets high in saturated and polyunsaturated fats, and polyunsaturated fat plus a-tocopherol supplemention (400 IU per day). On day 14 there was a significant difference in adduct levels between diets with saturated fats giving higher levels than polyunsaturated fats but this effect had disappeared by day 20 indicating that there is a relatively rapid adjustment to the effects on DNA damage of changes in dietary fat. a-Tocopherol showed a small benefit by day 20. Five females participated in the PUFA study and had higher mean adduct levels than men but there was no correlation with hormonal status. Overall, PUFA had a limited beneficial effect on M 1 dG levels that warrants further investigation.