Determination of malondialdehyde-induced DNA damage in human tissues using an immunoslot blot assay.
Malondialdehyde (MDA) is a product of lipid peroxidation and prostaglandin biosynthesis. It is mutagenic and carcinogenic and the major adduct formed by reaction with DNA, a highly fluorescent pyrimidopurinone (M1-dG), has been detected in healthy human liver and leukocyte DNA. Analytical methods used so far for the detection of M1-dG have not been applied to a large number of individuals or variety of samples. Often, only a few microg of DNA from human tissues are available for analysis and a very sensitive assay is needed in order to detect background levels of M1-dG in very small amounts of DNA. In this paper, the development of an immunoslot blot (ISB) assay for the measurement of MI-dG in 1 microg 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 and levels of 5.6-9.5 (n = 8) and 3.1-64.3 (n = 42) of M1-dG per 10(8) normal bases were detected in white blood cell and gastric biopsy DNA, respectively. Results on four human samples were compared with those obtained using an HPLC/32P-post-labelling (HPLC/PPL) method previously developed and indicated a high correlation between M1-dG levels measured by the two assays. The advantages of ISB over other assays including HPLC/PPL, such as the possibility of analysing 1 microg DNA/sample and 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 samples in biomonitoring studies.
Leuratti
C
C
MRC Toxicology Unit, University of Leicester, UK. c119@le.ac.uk
Singh
R
R
Lagneau
C
C
Farmer
P B
PB
Plastaras
J P
JP
Marnett
L J
LJ
Shuker
D E
DE
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000187:drug effects* / Q000378:metabolism; D018736:DNA Adducts / Q000032:analysis*; D004249:DNA Damage; D006801:Humans; D015151:Immunoblotting; D008315:Malondialdehyde / Q000378:metabolism / Q000633:toxicity*; D051379:Mice
D018736:DNA Adducts; D008315:Malondialdehyde; D004247:DNA