Measurements by filter elution of DNA single- and double-strand breaks in rat hepatocytes: effects of nitrosamines and gamma-irradiation
This work presents a filter elution method for measuring DNA single- and double-strand breaks in primary rat hepatocytes without radioactive labeling of DNA. Researchers have studied the effects of a series of nitrosamines and of gamma-irradiation on DNA single- and double-strand break induction. The repair of DNA single-strand breaks in the hepatocytes was measured after treatment with /sup 60/Co, 1-methyl-1-nitrosourea, and N-nitrosodimethylamine. The hepatocytes were isolated by ethylene glycol-bis(beta-aminoethyl ether)-N,N'-tetra acetic acid-collagenase perfusion and had a mean viability of 91 +/- 4% (S.D.). The isolated cells were treated for varying lengths of time with nitrosamines in suspension culture in L-15 medium containing 10% fetal bovine serum. After treatment, the cells were chilled, loaded onto 2 micrometers polycarbonate filters, and lysed in a 2% sodium dodecyl sulfate-proteinase K solution, pH 9.6. The DNA was eluted from the filter at either native or denaturing pH with fractions collected every 3 hr. The quantity of DNA in each fraction was determined by measuring the fluorescent product formed between it and diaminobenzoic acid after ethanol-sodium acetate precipitation and trapping of the DNA on 0.2-micrometer polycarbonate filters. The results show that the carcinogens, N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosodipropylamine, N-nitrosodibutylamine, and 1-nitrosopiperidine all made dose- and time-related increases in the number of single-strand breaks in rat hepatocytes. N-Nitrosodiphenylamine produced small numbers of single-strand breaks. No double-strand breaks were formed by any of the nitrosamines. Single-strand breaks induced by N-nitrosodimethylamine were repaired very slowly relative to repair of either gamma-ray of 1-methyl-1-nitrosourea-induced single-strand breaks. This system has many advantages for studying carcinogen metabolism and DNA damage in hepatocytes, one of the major target cells for many carcinogens.
- Research Organization:
- Merck Institute for Therapeutic Research, West Point, Pennsylvania
- OSTI ID:
- 7067529
- Journal Information:
- Cancer Res.; (United States), Journal Name: Cancer Res.; (United States) Vol. 42:7; ISSN CNREA
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
AMINES
ANIMAL CELLS
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOLOGICAL EFFECTS
BIOLOGICAL RADIATION EFFECTS
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
BODY
CARCINOGENS
CHEMICAL ANALYSIS
COBALT 60
COBALT ISOTOPES
DIGESTIVE SYSTEM
DNA
ELECTROMAGNETIC RADIATION
FILTRATION
GAMMA RADIATION
GENETIC EFFECTS
GENETIC RADIATION EFFECTS
GLANDS
INTERMEDIATE MASS NUCLEI
INTERNAL CONVERSION RADIOISOTOPES
IONIZING RADIATIONS
ISOMERIC TRANSITION ISOTOPES
ISOTOPES
LIVER
MAMMALS
MEASURING METHODS
MINUTES LIVING RADIOISOTOPES
NITROSO COMPOUNDS
NUCLEI
NUCLEIC ACIDS
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
PROMPT GAMMA RADIATION
QUANTITATIVE CHEMICAL ANALYSIS
RADIATION EFFECTS
RADIATIONS
RADIOISOTOPES
RATS
RECOVERY
REPAIR
RODENTS
SEPARATION PROCESSES
VERTEBRATES
YEARS LIVING RADIOISOTOPES