An improved (32)P-postlabeling assay for the sensitive detection of 8-oxodeoxyguanosine in tissue DNA.
To use oxidative DNA lesions as biomarkers of exposure and disease in the human scenario, the assay should not only be sensitive but also applicable to small quantities of DNA that is obtained from human tissue biopsies. Previously, we reported a nonenrichment (32)P-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]. Here we report a substantial improvement of the assay by enriching 8-oxo-dGp by TLC prior to (32)P-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, (32)P-labeled, resolved by TLC, and quantitated. The 8-oxo-dG signal was found to increase linearly with increasing amount of DNA. Its recovery was found to be 55-70%, as determined by using synthetic 8-oxo-dGp. Since the modified assay allowed using larger quantities of DNA and higher specific activity [gamma-(32)P]ATP than those used in the nonenrichment procedure, the signal-to-noise ratio increased to 30-100:1 from 3-5:1. The assay has a sensitivity of detection of <1 8-oxo-dG/10(7) nucleotides 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-old 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, respectively.
Gupta
R C
RC
Preventive Medicine and Environmental Health, 354 Health Sciences Research Building, University of Kentucky Medical Center, Lexington, KY 40536-0305, USA. rcgupta@pop.uky.edu
Arif
J M
JM
D000818:Animals; D015415:Biomarkers / Q000032:analysis*; D004249:DNA Damage; D010084:Oxidation-Reduction; D010761:Phosphorus Radioisotopes; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D014018:Tissue Distribution
D015415:Biomarkers; D010761:Phosphorus Radioisotopes