A sensitive immunoslot-blot assay for detection of malondialdehyde-deoxyguanosine in human DNA.
As part of a large programme on food risk assessment, we have become Interested in the endogenous production of genotoxic agents from dietary precursors. Malondialdehyde (MDA), a product of lipid peroxidation and prostaglandin biosynthesis, is mutagenic in bacterial and mammalian systems. MDA reacts with DNA, and the major adduct (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 large numbers of individuals or a large variety of samples. Often, only a few micrograms of DNA from human tissues are available for analysis, and a very sensitive assay is needed to detect background levels of M1-dG in very small amounts of DNA. In this paper, we describe the development of an immunoslot-blot (ISB) assay for the measurement of M1-dG in 1 microgram of DNA. The limit of detection of the assay is about 5 adducts per 10(8) bases. The advantages of ISB over other assays for DNA adduct detection, such as the possibility of analysing 1 microgram DNA per sample and the fact that it is less time-consuming and laborious, mean that it can be more easily used for routine analysis of large numbers of samples in biomonitoring. A series of human samples was analysed, and levels of 0.3-6.43 M1-dG per 10(7) normal bases were detected in 42 gastric biopsy samples and 0.7-16.65 M1-dG per 10(7) normal bases in 28 samples of leukocyte DNA. In an initial study in five human volunteers on standardized diets, the levels of M1-dG in leukocyte DNA changed in relation to meat, vegetable and tea intake.
Leuratti
C
C
MRC Toxicology Unit, University of Leicester, United Kingdom.
Singh
R
R
Deag
E J
EJ
Griech
E
E
Hughes
R
R
Bingham
S A
SA
Plastaras
J P
JP
Marnett
L J
LJ
Shuker
D E
DE
D015415:Biomarkers; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000032:analysis*; D018736:DNA Adducts / Q000032:analysis* / Q000097:blood; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000097:blood; D004032:Diet; D005753:Gastric Mucosa / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D007962:Leukocytes / Q000378:metabolism; D008956:Models, Chemical; D012680:Sensitivity and Specificity
D015415:Biomarkers; D018736:DNA Adducts; C089188:3-(2'-deoxyribofuranosyl)pyrimido(1,2-a)purin-10(3H)-one; D004247:DNA; D003849:Deoxyguanosine