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Title: /sup 32/P-postlabeling assay for carcinogen-DNA adducts: nuclease P/sub 1/-mediated enhancement of its sensitivity and applications

Abstract

Exceedingly sensitive assays are required for the detection of DNA adducts formed in humans exposed to low levels of environmental genotoxicants and therapeutic drugs. A /sup 32/P-postlabeling procedure for detection and quantitation of aromatic carcinogen-DNA lesions with a sensitivity limit of 1 adduct in 10/sup 7/ to 10/sup 8/ nucleotides has been described previously. In the standard procedure, DNA is enzymatically digested to 3'-phosphorylated normal and adducted mononucleotides, which are /sup 32/P-labeled at their 5'-hydroxyl groups by T4 polynucleotide kinase-catalyzed (/sup 32/P) phosphate transfer from (..gamma..-/sup 32/P)ATP. /sup 32/P-labeled derivatives are resolved by TLC, detected by autoradiography, and quantitated by counting. This assay has been recently utilized for the determination and partial characterization of DNA adducts formed in somatic and reproductive tissues of rats given the clinically used anticancer drug, mitomycin C. The drug exhibits similar levels of covalent binding to DNA in most tissues. Further studies have revealed that adducted nucleotides are primarily guanine derivatives that are resistant to 3'-dephosphorylation by Penicillium citrinum nuclease P/sub 1/. The latter observation has been utilized to enhance the /sup 32/P-assay's sensitivity to 1 adduct in 10/sup 10/ nucleotides for a 10-..mu..g DNA sample by postincubation of DNA digests with nuclease P/sup 1/more » before /sup 32/P-labeling. The new assay has also shown utility in the analysis of very low levels of age- and tissue-related DNA modifications, which might arise from dietary or endogenous compounds, in untreated rats and humans.« less

Authors:
;
Publication Date:
Research Org.:
Texas Medical Center, Houston (USA)
OSTI Identifier:
5068033
Resource Type:
Journal Article
Journal Name:
Environ. Health Perspect.; (United States)
Additional Journal Information:
Journal Volume: 76
Country of Publication:
United States
Language:
English
Subject:
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.; 62 RADIOLOGY AND NUCLEAR MEDICINE; DIMETHYLBENZANTHRACENE; BIOLOGICAL EFFECTS; DNA ADDUCTS; RADIOASSAY; AUTORADIOGRAPHY; CARCINOGENS; NUCLEASES; PHOSPHORUS 32; RATS; THIN-LAYER CHROMATOGRAPHY; ADDUCTS; ANIMALS; AROMATICS; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; CHROMATOGRAPHY; CONDENSED AROMATICS; DAYS LIVING RADIOISOTOPES; ENZYMES; ESTERASES; HYDROLASES; ISOTOPES; LIGHT NUCLEI; MAMMALS; NUCLEI; ODD-ODD NUCLEI; ORGANIC COMPOUNDS; PHOSPHODIESTERASES; PHOSPHORUS ISOTOPES; RADIOISOTOPES; RODENTS; SEPARATION PROCESSES; VERTEBRATES; 560300* - Chemicals Metabolism & Toxicology; 550601 - Medicine- Unsealed Radionuclides in Diagnostics

Citation Formats

Reddy, M V, and Randerath, K. /sup 32/P-postlabeling assay for carcinogen-DNA adducts: nuclease P/sub 1/-mediated enhancement of its sensitivity and applications. United States: N. p., 1987. Web. doi:10.2307/3430463.
Reddy, M V, & Randerath, K. /sup 32/P-postlabeling assay for carcinogen-DNA adducts: nuclease P/sub 1/-mediated enhancement of its sensitivity and applications. United States. doi:10.2307/3430463.
Reddy, M V, and Randerath, K. Tue . "/sup 32/P-postlabeling assay for carcinogen-DNA adducts: nuclease P/sub 1/-mediated enhancement of its sensitivity and applications". United States. doi:10.2307/3430463.
@article{osti_5068033,
title = {/sup 32/P-postlabeling assay for carcinogen-DNA adducts: nuclease P/sub 1/-mediated enhancement of its sensitivity and applications},
author = {Reddy, M V and Randerath, K},
abstractNote = {Exceedingly sensitive assays are required for the detection of DNA adducts formed in humans exposed to low levels of environmental genotoxicants and therapeutic drugs. A /sup 32/P-postlabeling procedure for detection and quantitation of aromatic carcinogen-DNA lesions with a sensitivity limit of 1 adduct in 10/sup 7/ to 10/sup 8/ nucleotides has been described previously. In the standard procedure, DNA is enzymatically digested to 3'-phosphorylated normal and adducted mononucleotides, which are /sup 32/P-labeled at their 5'-hydroxyl groups by T4 polynucleotide kinase-catalyzed (/sup 32/P) phosphate transfer from (..gamma..-/sup 32/P)ATP. /sup 32/P-labeled derivatives are resolved by TLC, detected by autoradiography, and quantitated by counting. This assay has been recently utilized for the determination and partial characterization of DNA adducts formed in somatic and reproductive tissues of rats given the clinically used anticancer drug, mitomycin C. The drug exhibits similar levels of covalent binding to DNA in most tissues. Further studies have revealed that adducted nucleotides are primarily guanine derivatives that are resistant to 3'-dephosphorylation by Penicillium citrinum nuclease P/sub 1/. The latter observation has been utilized to enhance the /sup 32/P-assay's sensitivity to 1 adduct in 10/sup 10/ nucleotides for a 10-..mu..g DNA sample by postincubation of DNA digests with nuclease P/sup 1/ before /sup 32/P-labeling. The new assay has also shown utility in the analysis of very low levels of age- and tissue-related DNA modifications, which might arise from dietary or endogenous compounds, in untreated rats and humans.},
doi = {10.2307/3430463},
journal = {Environ. Health Perspect.; (United States)},
number = ,
volume = 76,
place = {United States},
year = {1987},
month = {12}
}