Autoradiographic detection of diphtheria toxin resistant mutants in human diploid fibroblasts

Journal Article · Tue Jan 01 04:00:00 EST 1985 · Environ. Mutagen.; (United States)

DOI:https://doi.org/10.1002/em.2860070502· OSTI ID:6379820

Gupta, R S; Singh, B

An autoradiographic procedure for the detection of diphtheria toxin (DT) resistant (Dip/sub R/) mutants in human diploid fibroblast (HDF) cells has been developed. The assay is based on the observation that when HDFs from confluent cultures are seeded in medium containing 0.01 flocculating units/ml or higher concentration of DT, protein synthesis in sensitive cells is severely inhibited by 4-6 hr. If at this or later time, a radiolabeled protein precursor (eg, /sup 3/H-leucine) is added to the culture, it is almost exclusively incorporated into the resistant cells, which are then readily identified by autoradiography. These studies provide strong evidence that the labeled cells identified by autoradiography are bona fide Dip/sub R/ mutants. The detection of Dip/sub R/ cells by autoradiography is apparently not affected by the presence of the sensitive cells in the mixtures. The spontaneous frequency of Dip/sub R/ cells in HDFs has been found to be in the range of 1-5 x 10/sup -6/, and this increases in a dose dependent manner upon treatment with the mutagen ethyl methanesulfonate. These results indicate that the autoradiographic assay could be used for quantitative mutagenesis. Since the autoradiographic assay does not depend on cell division, it may prove useful in estimating the incidence of pre-existing mutations in cell populations that either do not divide or have very limited growth potential (eg, lymphocytes, muscle cells, neurons, senescent fibroblasts, etc.).

• Research Organization: McMaster Univ., Hamilton, Ontario
• OSTI ID: 6379820
• Journal Information: Environ. Mutagen.; (United States), Journal Name: Environ. Mutagen.; (United States) Vol. 7:5; ISSN ENMUD
• Country of Publication: United States
• Language: English

Related Subjects

550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
560301 -- Chemicals Metabolism & Toxicology-- Cells-- (-1987)
62 RADIOLOGY AND NUCLEAR MEDICINE
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
AMINO ACIDS
ANIMAL CELLS
ANTIGENS
AUTORADIOGRAPHY
BACTERIAL DISEASES
BIOSYNTHESIS
CARBOXYLIC ACIDS
CONNECTIVE TISSUE CELLS
DIPHTHERIA
DISEASES
EMS
ESTERS
FIBROBLASTS
INFECTIOUS DISEASES
LABELLED COMPOUNDS
LEUCINE
MATERIALS
MUTAGEN SCREENING
MUTAGENS
MUTANTS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PROTEINS
SCREENING
SOMATIC CELLS
SULFONIC ACID ESTERS
SYNTHESIS
TOXIC MATERIALS
TOXINS
TRITIUM COMPOUNDS