An improved P-32-postlabeling assay for the sensitive detection of 8-oxodeoxyguanosine in tissue DNA

Citation
Rc. Gupta et Jm. Arif, An improved P-32-postlabeling assay for the sensitive detection of 8-oxodeoxyguanosine in tissue DNA, CHEM RES T, 14(8), 2001, pp. 951-957
Citations number
33
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Pharmacology & Toxicology
Journal title
CHEMICAL RESEARCH IN TOXICOLOGY
ISSN journal
0893-228X → ACNP
Volume
14
Issue
8
Year of publication
2001
Pages
951 - 957
Database
ISI
SICI code
0893-228X(200108)14:8<951:AIPAFT>2.0.ZU;2-1
Abstract
To use oxidative DNA lesions as biomarkers of exposure and disease in the h uman scenario, the assay should not only be sensitive but also applicable t o small quantities of DNA that is obtained from human tissue biopsies. Prev iously, we reported a nonenrichment P-32-postlabeling method for measuring the oxidative DNA lesion, 8-oxo-7,8-dihydro-2 ' -deoxyguanosine (8-oxo-dG) [Devanaboyina, U., and Gupta, R. C. (1996) Carcinogenesis 17, 917-924]. Her e we report a substantial improvement of the assay by enriching 8-oxo-dGp b y TLC prior to P-32-labeling. Thus, unmodified nucleotides were removed by 1-directional polyethyleneimine (PEI)-cellulose TLC in 0.2 M formic acid. 8 -Oxo-dGp, retained close to the origin, was eluted in 2 M triethylammonium acetate (pH 7.0), lyophilized, P-32-labeled, resolved by TLC, and quantitat ed. The 8-oxo-dG signal was found to increase linearly with increasing amou nt of DNA. Its recovery was found to be 55-70%, as determined by using synt hetic 8-oxo-dGp. Since the modified assay allowed using larger quantities o f DNA and higher specific activity [gamma-P-32]ATP than those used in the n onenrichment procedure, the signal-to-noise ratio increased to 30-100:1 fro m 3-5:1. The assay has a sensitivity of detection of < 18-oxo-dG/10(7) nucl eotides using submicrogram to microgram DNA. The 8-oxo-dG levels (mean +/- SE; n = 5) in the liver, lung, heart, bladder, and trachea DNA of 3 month-o ld female Sprague-Dawley rats were found to be 1.05 +/- 0.24, 0.97 +/- 0.09 , 0.75 +/- 0.15, 0.79 +/- 0.15, and 1.17 +/- 0.28 per 10(6) nucleotides, re spectively.