Nick translation - a new assay for monitoring DNA damage and repair in cultured human fibroblasts
An in vitro assay has been developed to detect DNA damage and repair following chemical treatment of human diploid fibroblasts. DNA damage is measured by following the Escherichia coli DNA polymerase I-catalyzed incorporation of radiolabeled deoxycytidine triphosphate (dCTP) into the DNA of lysolecithin-permeabilized cells. DNA strand breaks with free 3' OH termini serve as template sites for incorporation, and decrease of this incorporation with time, following removal of the test chemical, indicates loss (repair) of initial damage. Inhibition of the DNA excision repair process by the addition of the repair inhibitors arabinofuranosyl cytosine (ara-C) and hydroxyurea (HU) during the incubation period gives rise to an increased number of template sites, manifesting itself in increased incorporation and indicating the induction of long-patch excision repair. Results presented demonstrate that all 14 direct-acting carcinogens tested and 8 of 14 carcinogens requiring metabolic activation give positive indication of DNA damage, repair, or both. Eleven of 14 noncarcinogens tested were scored as negative, the other 3 having previously been shown to interact with cellular DNA. This assay is shown to have predictive capability at least equal to that of UDS assays but to allow a broader spectrum of genotoxic effects to be analyzed.
- Research Organization:
- Stauffer Chemical Co., Farmington, CT
- OSTI ID:
- 6525866
- Journal Information:
- Environ. Mutagen.; (United States), Journal Name: Environ. Mutagen.; (United States) Vol. 7:3; ISSN ENMUD
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
560301* -- Chemicals Metabolism & Toxicology-- Cells-- (-1987)
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
AFLATOXIN
ANIMAL CELLS
ANIMALS
ANTIGENS
AROMATICS
BACTERIA
BENZOPYRENE
BIOASSAY
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
CARCINOGEN SCREENING
CONDENSED AROMATICS
CONNECTIVE TISSUE CELLS
DNA POLYMERASES
DNA REPAIR
DNA REPLICATION
ELECTROMAGNETIC RADIATION
EMS
ENZYMES
ESCHERICHIA COLI
ESTERS
FIBROBLASTS
HYDROCARBONS
IN VITRO
LABELLED COMPOUNDS
MAMMALS
MAN
MATERIALS
METABOLIC ACTIVATION
METHYL METHANESULFONATE
MICROORGANISMS
MUTAGENS
NITROSO COMPOUNDS
NITROSOUREAS
NUCLEIC ACID REPLICATION
NUCLEOTIDYLTRANSFERASES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
PRIMATES
RADIATIONS
RECOVERY
REPAIR
SCREENING
SOMATIC CELLS
STRAND BREAKS
SULFONIC ACID ESTERS
TOXIC MATERIALS
TOXINS
TRANSFERASES
TRITIUM COMPOUNDS
ULTRAVIOLET RADIATION
VERTEBRATES