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

Journal Article · Tue Dec 01 04:00:00 EST 1987 · Environ. Health Perspect.; (United States)

DOI:https://doi.org/10.2307/3430463· OSTI ID:5068033

Reddy, M V; Randerath, K

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.

• Research Organization: Texas Medical Center, Houston (USA)
• OSTI ID: 5068033
• Journal Information: Environ. Health Perspect.; (United States), Journal Name: Environ. Health Perspect.; (United States) Vol. 76; ISSN EVHPA
• Country of Publication: United States
• Language: English

Related Subjects

550601 -- Medicine-- Unsealed Radionuclides in Diagnostics
560300* -- Chemicals Metabolism & Toxicology
62 RADIOLOGY AND NUCLEAR MEDICINE
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ADDUCTS
ANIMALS
AROMATICS
AUTORADIOGRAPHY
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOLOGICAL EFFECTS
CARCINOGENS
CHROMATOGRAPHY
CONDENSED AROMATICS
DAYS LIVING RADIOISOTOPES
DIMETHYLBENZANTHRACENE
DNA ADDUCTS
ENZYMES
ESTERASES
HYDROLASES
ISOTOPES
LIGHT NUCLEI
MAMMALS
NUCLEASES
NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHODIESTERASES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
RADIOASSAY
RADIOISOTOPES
RATS
RODENTS
SEPARATION PROCESSES
THIN-LAYER CHROMATOGRAPHY
VERTEBRATES