32P-Post-labelling method improvements for aromatic compound-related molecular epidemiology studies

• DOI: 10.1093/mutage/gem030
• Publication Date: 2007-08-24T00:14:23Z
• Authors: A. Munnia; F. Saletta; A. Allione; S. Piro; M. Confortini; G. Matullo; M. Peluso
• Keywords: DNA adduct; Sample Preparation

The (32)P-post-labelling assay has emerged as a major tool for detecting bulky DNA adducts in subjects exposed to carcinogens, especially aromatic compounds. However, the protocol still requires use of high amounts radioactivity, i.e. 25-50 muCi per sample, an obstacle that limits its large studies. characterization measured is also limited. Methodological improvements and increased adduct are necessary make this capable achieving higher throughput. A new was tested ensure efficient hydrolysis reduce radioactive material obtain chromatography resolution. Different systems based on high-urea or ammonium hydroxide were employed characterize being measured. Improvements by re-analysing group police officers urban residents Genoa, Italy. analysis carcinogen-modified standards included study qualitative quantitative comparison. An digestion obtained using method involving micrococcal nuclease mixture two spleen phosphodiesterases at fixed concentrations. 72% reduction amount radioactivity used labelling achieved respect non-modified without loss sensitivity. improved resolution reducing volume sample be spotted chromatogram. Lower spotting can decrease diffusion formation unresolved spots thin-layer plate. output produced single batch carrier-free [gamma-(32)P]ATP about 3.5-fold. complex pattern observed leukocytes both isopropanol-ammonium systems, techniques effective detection adducts. above observations indicate likely have been induced