10 Pages

Induction of Diphtheria Toxin-resistant Cells by Mutagen-carcinogens

ByMasaaki Terada, Michie Nakayasu, Hiromi Sakamoto, Fumiko Nakasato, Takashi Sugimura

A mutation assay system with Chinese hamster lung (CHL) cells in culture has been established using diphtheria toxin resistance as a phenotypic selection marker. The results of series of studies on this mutation assay system are summarized and presented. Dose-dependent increase in the number of diphtheria toxin-resistant (DTr) cells was observed when the cells were exposed to a mutagen-carcinogen and then incubated in fresh medium for an expression period of 7 to 8 days. After exposure to ethyl methanesulfonate, the number of DTr cells was much higher than the number of thioguanine-or ouabain-resistant cells. ADP-ribosylation of elongation factor 2 (EF-2) catalyzed by diphtheria toxin was measured in cell-free extracts from the parent cells and 17 DTr cells, including 6 spontaneous DTr cells and 11 DTr cells induced by mutagens; the numbers of ADP-ribose molecules transfer to EF-2 in extracts of mutant cells were less than 1 % of that in extract of the parent cells.

Various mutagen-carcinogens, including heterocyclic amines isolated from pyrolysates of amino acids, proteins, and broiled fish, have been assayed with this mutation assay system in the presence or absence of a metabolic activation system and the results of these studies are also presented.

In addition, a method to detect DTr cells in situ has been developed using autoradiographic method. The potential usefulness of this method for detection of DTr cells with no proliferative capacity to form colony and for analysis of cellular events occurring after exposure of the cells to mutagen-carcinogens is discussed.