/sup 32/P-postlabeling analysis of non-radioactive aromatic carcinogen-DNA adducts
- Texas Medical Center, Houston (USA)
A newly developed enzymatic /sup 32/P-postlabeling method was applied to the analysis of DNA's containing non-radioactive arylamine, arylamide, and polycyclic aromatic hydrocarbon adducts. DNA reacted in vitro with N-hydroxy-2-amino-fluorene, N-acetoxy-2-acetylaminofluorene, and 7..beta..,8..cap alpha..-dihydroxy-9..cap alpha.., 10..cap alpha..-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, respectively, as well as DNA preparations from the livers of rats treated with N-hydroxy-2-acetylaminofluorene and benzo(a)pyrene, respectively, were enzymatically digested to deoxyribonucleoside 3'-monophosphates, which were then converted to (5'-/sup 32/P)deoxyribonucleoside 3',5'-bisphosphates by T4 polynucleotide kinase-catalyzed (/sup 32/P)phosphate transfer from (..gamma..-/sup 32/P)ATP. The /sup 32/P-labeled nucleotides were resolved by anion-exchange t.l.c. on polyethyleneimine-cellulose and detected by autoradiography. Aromatic adduct nucleotides were found to be retained at the origin in aqueous electrolyte solutions but to migrate as distinct spots in solvents containing 7-8.5 M urea. Advantage was taken of this observation to remove /sup 32/P-labeled normal DNA nucleotides from adduct nucleotides. This purification enabled the detection of a single adduct in 10/sup 7/-10/sup 8/ normal nucleotides. The method appears applicable to the ultrasensitive detection of a large number of carcinogen-DNA adducts of diverse structure without requiring radioactive carcinogens or specific antibodies.
- OSTI ID:
- 6120642
- Journal Information:
- Carcinogenesis (N.Y.); (United States), Vol. 3:9
- Country of Publication:
- United States
- Language:
- English
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CARCINOGENS
DETECTION
TRACER TECHNIQUES
ADDUCTS
BENZOPYRENE
CALVES
DNA
IN VITRO
IN VIVO
LABELLING
LIVER
PHOSPHORUS 32
RADIOENZYMATIC ASSAY
RATS
THYMUS
ANIMALS
AROMATICS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BODY
CATTLE
CONDENSED AROMATICS
DAYS LIVING RADIOISOTOPES
DIGESTIVE SYSTEM
DOMESTIC ANIMALS
GLANDS
HYDROCARBONS
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
LYMPHATIC SYSTEM
MAMMALS
NUCLEI
NUCLEIC ACIDS
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANS
PHOSPHORUS ISOTOPES
RADIOISOTOPES
RODENTS
RUMINANTS
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550901* - Pathology- Tracer Techniques