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), Vol. 7:3
- Country of Publication:
- United States
- Language:
- English
Similar Records
Sensitivity of excision repair in normal human, xeroderma pigmentosum variant and Cockayne's syndrome fibroblasts to inhibition by cytosine arabinoside
Use of metabolic inhibitors to compare the excision repair of pyrimidine dimers and nondimer DNA damages in human skin fibroblasts exposed to 254-nm and sunlamp-produced > 310-nm ultraviolet radiation
Related Subjects
DNA REPAIR
BIOASSAY
STRAND BREAKS
AFLATOXIN
BENZOPYRENE
CARCINOGEN SCREENING
DNA POLYMERASES
DNA REPLICATION
EMS
ESCHERICHIA COLI
FIBROBLASTS
IN VITRO
MAN
METABOLIC ACTIVATION
METHYL METHANESULFONATE
NITROSO COMPOUNDS
NITROSOUREAS
TRITIUM COMPOUNDS
ULTRAVIOLET RADIATION
ANIMAL CELLS
ANIMALS
ANTIGENS
AROMATICS
BACTERIA
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
CONDENSED AROMATICS
CONNECTIVE TISSUE CELLS
ELECTROMAGNETIC RADIATION
ENZYMES
ESTERS
HYDROCARBONS
LABELLED COMPOUNDS
MAMMALS
MATERIALS
MICROORGANISMS
MUTAGENS
NUCLEIC ACID REPLICATION
NUCLEOTIDYLTRANSFERASES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
PRIMATES
RADIATIONS
RECOVERY
REPAIR
SCREENING
SOMATIC CELLS
SULFONIC ACID ESTERS
TOXIC MATERIALS
TOXINS
TRANSFERASES
VERTEBRATES
560301* - Chemicals Metabolism & Toxicology- Cells- (-1987)
560121 - Radiation Effects on Cells- External Source- (-1987)