Genetics of somatic mammalian cells. IX. Quantitation of mutagenesis by physical and chemical agents

Abstract

The system previously described for inducing single gene mutations in Chinese hamster cells has been extended to produce additional auxotrophic mutants. An improved method for quantitating the efficiency of single gene mutation to specific auxotrophies has been developed. Mutagenesis in the forward direction has been measured after treatment of these cells with ethyl methanesulfonate, N-methyl-"-nitro-N-nitrosoguanidine, hydroxylamine, a n acridine mustard , caffeine and ultraviolet-and X-irradiation. For each agent, the single cell survival curve and the efficiency of chromatid breakage and rearrangement were measured. Similar measurements were also carried out with a water-soluble carcinogen N-nitrosomethylurea, which was shown to be effective in producing auxotrophic, somatic mutations. These results offer promise of illuminating the relationships between cell killing, chromosomal aberration, single gene mutations and carcinogenesis produced by various agents. The methods described can be used in routine testing of drugs, food additives, and environmental pollutants for mutagenic action in mammalian cells in vitro.In preceding papers of this series, a system was described in which mutations to nutritional deficiency could be produced in Chinese hamster cells grown in vitro, and the corresponding clones isolated. The mutations so obtained are single-step; they have reversion frequencies in the neighborhood of to lo-* so that experiments with high resolving power are possible; the deficient mutants so far described are "nonleaky," i.e., they display no growth whatever in the absence of the required nutrilite, so permitting easy scoring of mutagenesis; and the chromosomal constitution of the original cell and its various mutants is satisfactorily constant with a near-diploid number. Mutagenesis by ethyl methanesulfonate (EMS) and N-methyl-"nitro-N nitrosoguanidine (MNNG) was described, which produced mutants with growth requirements for glycine (gly-) or inositol (ino-) and mutants requiring a combination of glycine, hypoxanthine, and thymidine (gly-+ hyp-+ thy-). Reverse mutants to prototrophy from proline-requiring forms were also produced with EMS and MNNG. Some quantitative data were presented for both forward and reverse mutation frequencies at various loci produced by the mutagens employed (Kao and Puck, J. CELL. PHYSIOL., 74: 245-258 '68). Chu and Malling ('68) confirmed the production of mutations by these chemical agents in Chinese hamster cells using other genetic markers.In the present paper the production of additional auxotrophic mutations is described; a larger variety of chemical mutagens is examined; and the action of X-and ultraviolet (UV) radiation is studied. These agents are compared quantitatively with each other for their effectiveness in producing forward mutations, cell killing, and chromosomal aberrations in Chinese hamster cells in vitro. In addition a beginning has been made in extending such studies to carcinogenic agents. MATERIALS AND METHODS(1) Cell culture. The previously d...

Specific suppressive factors produced by hybridomas derived from the fusion of enriched suppressor T cells and a T lymphoma cell line

“…The line L5178 was kindly supplied by Dr. Boyle in 1977. It was originally derived from a population of L5178Y that had been treated with the mutagen ICR-372 (11). The parent strain originated from a leukemic DBA/2 mouse (12).…”

“…The parent strain originated from a leukemic DBA/2 mouse (12). The mutant cells, which lack hypoxanthine/guanine phosphoribosyl transferase, cannot proliferate in hypoxanthine aminopterin thymidine medium (11). They express Thy-l.2 and the H-2 d alloantigens 4, 8, and 35.…”

Further Changes in Differentiation State Accompanying the Conversion of Chinese Hamster Cells to Fibroblastic Form by Dibutyryl Adenosine Cyclic 3′:5′-Monophosphate and Hormones

“…The properties of this cell line and procedures used in its cultivation have been reported (2,4 ,ug/ml of sodium ascorbate, and incubated for 16 hr. Collagen synthesis was measured by the method of Juva and Prockop (6) as adapted by Priest and Davies (7).…”

X-ray-induced cell killing and mutations in cultured human cell lines (xeroderma pigmentosum cells and HeLa S3 cells).

“…During the last decade, several mammalian cell systems suitable for the detection and quantitative evaluation of radiation-induced mutational events have been described by many workers (Kao and Puck 1969;Bridges and Huckle 1970;Arlett and Potter 1971;Chu 1971;Albertini and DeMars 1973;Knaap and Simons 1975;Cox and Masson 1976;Zeeland and Simons 1976;Thacker et al 1977). Among these Chinese hamster cell systems hold a special place because of a number of characteristics, such as relatively short generation time, high plating efficiency and small number of chromosomes In the previous studies Horikawa et al 1976), we have also established a sensitive forward mutation system, for detecting the mutations induced by radiations and chemical carcinogens or mutagens, using prototrophic CH-hai Cl 23 cells which were isolated from an original Chinese hamster hai cell line.…”