title: Optimizing immunoslot blot assays and application to low DNA adduct levels using an amplification approach.
abstract: 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 was examined systematically, and it was found that the major problems are in the DNA fragmentation step and the use of the manifold apparatus. Optimization was performed on both the malondialdehyde-deoxyguanosine (M(1)dG) adduct and the O(6)-carboxymethyl-deoxyguanosine (O(6)CMdG) adduct to demonstrate the applicability to other DNA adducts. Blood samples from the European Prospective Investigation on Cancer (EPIC) study (n = 162) were analyzed 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 versus vegetarian diet intervention (n = 6) were analyzed 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.
authors.affiliation: School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Liverpool L3 3AF, UK. s.a.moore@ljmu.ac.uk
authors.forename: Sharon A
authors.initials: SA
authors.lastname: Moore
authors.forename: Ourania
authors.initials: O
authors.lastname: Xeniou
authors.forename: Zuotao T
authors.initials: ZT
authors.lastname: Zeng
authors.forename: Emma
authors.initials: E
authors.lastname: Humphreys
authors.forename: Sally
authors.initials: S
authors.lastname: Burr
authors.forename: Elke
authors.initials: E
authors.lastname: Gottschalg
authors.forename: Sheila A
authors.initials: SA
authors.lastname: Bingham
authors.forename: David E G
authors.initials: DE
authors.lastname: Shuker
mesh_terms: D018736:DNA Adducts / Q000097:blood* / Q000737:chemistry* / Q000276:immunology; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000097:blood / Q000737:chemistry / Q000276:immunology; D004032:Diet; D005060:Europe; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D057230:Limit of Detection; D009369:Neoplasms / Q000737:chemistry; D011446:Prospective Studies
chemical_list: D018736:DNA Adducts; C107252:O(6)-(carboxymethyl)-2'-deoxyguanosine; C089188:3-(2'-deoxyribofuranosyl)pyrimido(1,2-a)purin-10(3H)-one; D003849:Deoxyguanosine