DETERMINATION OF MALONDIALDEHYDE-INDUCED DNA-DAMAGE IN HUMAN TISSUES USING AN IMMUNOSLOT BLOT ASSAY

Malondialdehyde (MDA) is a product of lipid peroxidation and prostagla ndin biosynthesis. It is mutagenic and carcinogenic and the major addu ct formed by reaction with DNA, a highly fluorescent pyrimidopurinone (M-1-dG), has been detected in healthy human liver and leukocyte DNA, Analytical methods used so far for the detection of M1-dG have not bee n applied to a large number of individuals or variety of samples. Ofte n, only a few mu g of DNA from human tissues are available for analysi s and a very sensitive assay is needed in order to detect background l evels of M1-dG in very small amounts of DNA, In this paper, the develo pment of an immunoslot blot (ISB) assay for the measurement of M-1-dG in 1 mu g of DNA is described. The limit of detection of the assay is 2.5 adducts per 10(8) bases, A series of human samples were analysed a nd levels of 5.6-9.5 (rt = 8) and 3.1-64.3 (n = 42) of M1-dG per 108 n ormal bases were detected in white blood cell and gastric biopsy DNA, respectively. Results on four human samples were compared with those o btained using an HPLC/P-32-post-labelling (HPLC/PPL) method previously developed and indicated a high correlation between M1-dG levels measu red by the two assays. The advantages of ISB over other assays includi ng HPLC/PPL, such as the possibility of analysing 1 mu g DNA/sample an d the fact that it is less time-consuming and laborious, means that it can be more easily used for routine analysis of a large number of sam ples in biomonitoring studies.