Ada G.A.C. Knaap

A mutational assay system for l5178y mouse lymphoma cells, using hypoxanthine-guanine-phosphoribosyl-transferase (hgprt) -deficiency as marker. The occurrence of a long expression time for mutations induced by x-rays and ems.

The development of a system for the detection of somatic cell mutation to hypoxanthine-guanine-phosphoribosyl-transferase (HGPRT) (EC 2.4.2.8) deficiency in L5178Y mouse lymphoma cells is described. The selection of mutant cells was not influenced by the concentration of the selective agent 6-thioguanine (6-TG). In addition, all the mutants selected, spontaneous as well as induced ones, showed a complete loss of HGPRT activity. In reconstruction experiments, in which mutant cells were mixed with wild-type cells, the recovery of mutant cells was only markedly influenced when wild-type cells were seeded in a cell density ten times higher than the one, 5-10(4) cells/ml, used in subsequent induction experiments. X-irradiation and treatment with ethyl methanesulfonate (EMS) increased in the mutation rate above the spontaneous background. A clear-cut dose-dependent mutagenic effect after exposure to X-rays was measured. The rate of induced mutations at the HGPRT locus in lymphoma cells was 1-3-10(-7) per R, as determined after exposures of 200, 300, 400, 500 and 600 R. The time the cells needed to express their mutations was much longer than 48 h. Further study of this phenomenon showed that the optimal expression time for TGr-resistant mutants in L5178Y cells was 6 to 7 days. No indication for a dose-dependent effect on the optimal expression of the mutants was found.

Molecular dosimetry of the chemical mutagen ethyl methanesulfonate. Quantitative comparison of mutation induction in escherichia coli, v79 chinese hamster cells and l5178y mouse lymphoma cells, and some cytological results in vitro and in vivo.

Molecular dosimetry studies were carried out to measure the extent of binding of radio-labeled ethyl groups to the DNA of Escherichia coli, V79 Chinese hamster cells and L5178Y mouse lymphoma cells treated with ethyl methanesulfonate (EMS). The results show that (1) the amount of ethylation of the DNA is similar in these cells when treatment conditions are identical, (2) the relationship between dose to DNA (ethylations per nucleotide) versus exposure (mM applied concentration) is non-linear in the sense that less alkylation of the DNA is observed at the higher exposures than would be predicted on the basis of proportionality between dose to DNA and exposure, and (3) the non-linearity of the genetic response in the bacterial cells is not reflected in a non-linearity of the alkylation of the DNA in those cells. Quantitative comparison of the frequencies of gene mutations in the various systems shows that the mutation frequency per unit of DNA alkylation is heterogeneous among the mammalian cell systems and that the frequencies observed in the bacterial cells fall within the range observed with mammalian cells. Alkylation of the DNA in the bone marrow, testis and liver of Swiss random-bred mice was also measured. The results support the conclusion that the distribution of the compound to the various tissues is rapid and probably uniform. Quantitative assessment of the cytological data (micronuclei, sister-chromatid exchanges, etc.) on the basis of dose was not as useful because of the low efficiency of EMS for inducing cytologically observable damage.

Mutagenicity of hycanthone in Drosophila melanogaster.

Abstract The schistosomicidal agent hycanthone was tested for mutagenicity in Drosophila melanogaster . The compound was administered either by injection into adult males or by larval feeding. The following types of genetic damage were measured: (1) complete and mosaic sex-linked recessive lethal mutations; (2) II–III translocations; and (3) dominant lethals. In postmeiotic germ cells, especially in late spermatids, a pronounced increase was found in the frequency of sex-linked recessive lethals, both completes and mosaics. By contrast, translocations and dominant lethals were not induced.

Mutagenecity tests with captan and folpet in Drosophila melanogaster

Abstract Captan (N-(trichloromethylthio)-3a,4,7,7a-tetrahydrophthalimide) and folpet (N-(trichloromethylthio)-phthalimide), two closely related fungicides, were tester for mutagenicity in Drosophila melanogaster. The compounds were administered either by injection into adult males or by feeding to larvae. The following types of genetic damage were measured: (1) complete and mosaic sex-linked recessive lethal mutations, (2) II–III translocations, and (3) dominant lethals. In some of the sex-linked lethal tests a slight increase in mutation rate was obtained, but this could not be confirmed in replicate experiments. It is concluded that in the system used the mutagenicity of captan and folpet could not be demonstrated, although a very weak effect cannot be excluded.

Comparison of the mutagenic potency of 2-chloroethanol, 2-bromoethanol, 1,2-epoxybutane, epichlorohydrin and glycidaldehyde in Klebsiella pneumoniae, Drosophila melanogaster and L5178Y mouse lymphoma cells

A series of 2 haloethanols and 3 epoxides was investigated in 3 mutagenicity test systems, namely (1) the fluctuation test in Klebsiella pneumoniae, (2) the sex-linked recessive lethal test in Drosophila melanogaster, and (3) the HGPRT test with L5178Y mouse lymphoma cells. The order of mutagenic potency was, in Klebsiella: glycidaldehyde greater than 2-bromoethanol = epichlorohydrin greater than 1,2-epoxybutane greater than 2-chloroethanol; in Drosophila: glycidaldehyde = epichlorohydrin greater than 1,2-epoxybutane; in mouse lymphoma cells: epichlorohydrin greater than 1,2-epoxybutane. The haloethanols were non-mutagenic in Drosophila. 2-Chloroethanol and glycidaldehyde were negative in mouse lymphoma cells. The high mutagenic potency of epichlorohydrin as compared with 1,2-epoxybutane was consistent in all systems, and with published data.

Effects of ethionine on the replicational fidelity in V79 Chinese hamster cells

The possibility that ethionine, the ethyl analog of methionine and potent liver carcinogen, exerts its action by blocking the methylation of DNA and thereby rendering the post-replicative methylation instructed error-avoidance system inoperative was investigated. While the results are not directly supportive for the existence of such a repair system in V79 Chinese hamster cells, effects of ethionine were found. Following the exposure of ethionine-treated cells to EMS an increase in cell killing and a decrease in mutation induction was observed. The base analog, 6-hydroxyaminopurine, was shown to be clearly mutagenic in the mammalian cells and in the presence of ethinine a drastic decrease in mutant frequency was observed. Ethionine itself did not appear mutagenic over the entire dose range tested (1-1000 micrograms/ml).

Establishment of a dose-response relationship for reverse mutation at the hprt (hypoxanthine guanine phosphoribosyl transferase) locus in l5178y mouse lymphoma cells.

L5178Y mouse lymphoma cells were treated with the mismatching agent 6-hydroxy-aminopurine (HAP), a base analogue known to produce forward and reverse mutations in bacteria. Mutants with the phenotype deficient in hypoxanthine guanine phosphoribosyl transferase (HPRT) were selected in 6-thoiguanine(TG)-containing medium and isolated. Reverse mutations to the HPRT-proficient phenotype occurred both spontaneously and after treatment with ethyl nitrosourea (ENU), which suggested that the initial HAP treatment had induced point mutations at the HPRT locus. Reconstruction experiments, in which a small number of wild-type cells together with different numbers of mutant cells were seeded in HAT medium, indicated that densities up to 10(6) cells per ml can be used for the selection of revertants. Optimal expression of induced revertants was obtained two days after treatment. The dose-response relationship for induction of reverse mutations by ENU appears not to deviate from linearity. The highest revertant frequency observed was 3.3 x 10(-5) at an ENU concentration of 1 mM. The spontaneous reversion frequency per generation -- based on 3 spontaneous revertants -- was estimated to be 1.3 x 10(-9). All revertants were indistinguishable from the parental wild-type line with respect to the activity as well as the electrophoretic mobility of HPRT.

Quantitative Molecular Dosimetry of Ethyl Methanesulfonate (EMS) in Several Genetic Test Systems

The increasing use of several different organisms (1, 2) to detect, verify, quantify, and extrapolate the potential mutagenic and carcinogenic effects of environmental chemicals (3) makes a critical evaluation of their sensitivity and reliability necessary. Of special interest is the determination to what extent the response to chemical mutagens is influenced by intrinsic cellular factors, such as chromosome structure and DNA content per cell, and also by metabolic and physiologic differences between test organisms or within strains of the same species (4). A quantitative comparison of the induced genetic effects first requires a precise determination of the actual dose at the target molecule, say, DNA. The reason for individual dose measurements in the different indicator organisms is that one cannot, a priori, assume that identical exposure concentrations of a chemical applied to the system will lead to identical dose at the DNA level. Instead, one expects that penetration and metabolism in general will influence the extent of reaction of the compound with DNA.

Low mutagenic activity of four hycanthone analogues in Drosophila melanogaster

Four structural analogues of the antischistosomal drug hycanthone, indicated as IA-3, IA-4, IA-5, and IA-6, were tested for their ability to induce sex-linked recessive lethal mutations in Drosophila melanogaster. The compounds were administered to adult male flies, by abdominal injection or by feeding. Although in some cases a slight enhancement of the mutation frequency was observed, the analogues tested are considerably less mutagenic in this system than hycanthone itself, the concentrations applied being approximately of equal molarity.

Ascertainment of the effect of differential growth rates of mutants on observed mutant frequencies in X-irradiated mammalian cells An indication for the occurrence of X-ray-induced untargeted mutagenesis

As it is not known to what extent differential growth rates of induced mutants lead to over- and under-representation of mutants in treated populations and thereby affect the determination of mutant frequencies, the mutation induction in X-irradiated L5178Y mouse lymphoma cells was determined via two methods. The first method involves the standard protocol which may suffer from the effect of differential growth rates, while the second method is based upon the fluctuation test in which the differential growth rates can be actually measured. It appeared that the standard protocol led to a mutant frequency that was similar to the mutant frequency determined in the fluctuation test. Therefore, the standard protocol appears to lead to only a minor under-estimation if any. Substantial heterogeneity in growth rates of induced mutants was observed, but the mutants with a selective advantage appear largely to compensate for the mutants that are lost because of selective disadvantage. It was calculated that the chance for isolating the same mutant twice from a treated population had been increased 2.2-fold because of the observed differential growth rates. Therefore, our data indicate that the standard protocol does not lead to serious errors in the determination of mutant frequencies and in the sampling of mutants. The fluctuation tests were also used to determine the spontaneous mutation frequency per cell per generation. The mutation rate appeared more than 10-fold enhanced in X-irradiated cells which may be attributed to the induction of a process of untargeted mutagenesis in mammalian cells.