Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. I. Application to genetic toxicological testing.

We have systematically evaluated the mouse lymphoma TK+/- leads to TK-/- mutagenesis assay to determine if this somatic-cell test system would be a useful addition to the routine screening battery already used in our laboratory for the detection of chemical mutagens. During these investigations we observed that, with certain modifications of the basic assay, mutagenicity data could be obtained in as little as 9 days once the relative cytotoxic properties of the test substance were known. By improving the culturing conditions, we were able to reduce the serum requirements by as much as 50--75% without appreciably altering either cell viability or the recovery of chemically-induced mutants. Phenotypic stability of test-derived trifluorothymidine resistant (TFTR) mutants was confirmed by demonstrating cross-resistance to bromodeoxyuridine and concomitant sensitivity to methotrexate (THMG) in TFTR cells grown for 20 generations under non-selective conditions. While reduced growth rates resulting from temporary cell-division delay in treated cells is probably not a contributing factor to the observed mutation frequencies, only TFTR colonies which formed large distinct colonies in the presence of trifluorothymidine were clearly phenotypically stable mutants when spontaneous mutants were isolated and verified. When a non-mutagen, a weak mutagen, and a well-established mutagen were compared at equitoxic doses under these modified conditions, clear quantitative differences were seen in the respective mutation frequencies induced by these 3 types of agents. With these technical modifications, we feel this assay is both reliable and amenable to the screening of diverse chemical compounds for point-mutational activity in a mammalian cell.     

Cytogenetic analysis of the L5178Y/TK+/− → TK−/− mouse lymphoma mutagenesis assay system

The L5178Y/TK^+/? → TK^?/? mouse lymphona mutagen assay, which allows selection of forward mutations at the autosomal thymidine kinase (TK) locus, uses a TK^+/? heterozygous cell line, TK^+/? 3.7.2C. Quantitation of colonies of mutant TK^?/? cells in the assay forms the basis for calculations of mutagenic potential of test compounds. We have evaluated the banded karyotypes of the parent TK^+/? heterozygous cell line, as well as homozygous TK^?/? mutants, in order to relate the genetic and morphological properties of mutant colonies. The parent cell line displays karyotype homogeneity, all cells containing normal mouse chromosomes, readily identifiable chromosome rearrangements, and cell line specific marker chromosomes. Mutant TK^?/? colonies of the TK^+/? 3.7.2C cell line form a bimodal frequency distribution of colony sizes for most mutagenic or carcinogenic test substances. Large-colony (λ) TK^?/? mutants with normal growth kinetics appear karyotypically identical within and among clones and with the TK^+/? parental cell line. In contrast, most slow-growing small-colony (σ) TK^?/? mutants have readily recognizable chromosome rearrangements involving chromosome 11, which contains the thymidine kinase gene locus. It is possible that the heritable differences in growth kinetics and resultant colony morphology in λ and σ mutants are related to the type of chromosomal damage sustained. Large-colony mutants receive minimal damage, possibly in the form of point mutations at the TK locus, while small-colony mutants receive damage to other genetic functions coordinately with loss of TK activity, implying gross insult to chromosomal material. It seems likely that λ and σ mutants result from 2 different mutational mechanisms that may be distinguished on the basis of mutant colony morphology.     

Activity of the plant flavanol quercetin in the mouse lymphoma L5178Y TK+/− mutation,DNA single-strand break,and Balb/c 3T3 chemical transformation assays

The activity of quercetin was investigated in (a) the L5178Y TK^+/− mutation assay system, using trifluorothymidine (TFT) as the selection agent; (b) the DNA single-strand break assay in L5178Y cells after the same treatment used for the mutation assay; and (c) the Balb/c 3T3 chemical transformation assay (foci method). Quercetin was active in the TK^+/− mutation assay, increasing the frequency of TFT-resistant colonies from a control value of 37 per 10⁶ viable cells to 355 per 10⁶ viable cells at 20 μg/ml. When S9 was present, the activity was decreased at each concentration tested. As the S9 concentration employed (mg/ml protein) was decreased, the induced mutant frequency increased. DNA single-strand breakage was observed without S9 at 10 μg/ml, using the alkaline elution technique; a maximal rate of elution was reached at 20 μg/ml. In the chemical transformation experiments, transformation just at the level of 0.05% significance (if both intermediate and typical transformed colonies were combined) was observed. The evidence is sufficiently strong that additional attention should be given to its role as a dietary caused of human cancer.     

Hamster hepatocyte-mediated activation of procarcinogens to mutagens in the L5178Y/TK mutation assay

Eight procarcinogens including three nitrosamines, three polycyclic hydrocarbons, and two aromatic amines were tested for mutagenic potential at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells co-cultivated with viable hamster hepatocytes. All eight chemicals produced substantial mutagenic activity as indicated by increased trifluorothymidine resistance in L5178Y cells treated in the presence of hepatocytes. Mutagenic responses to benzo[a]pyrene, 3-methyl-cholanthrene, N-nitrosodiethylamine, and N-nitrosodipropylamine first increased, then plateaued within the range of mutagen concentrations tested, while consistent dose-dependent increases in mutant frequencies were observed following 2-aminoanthracene, 2-aminofluorene, or N-nitrosodimethylamine treatments. The relatively flat portions of the mutant frequency curves for benzo[a]pyrene and 3-methylcholanthrene coincided with maximum chemical solubility as obvious from visible or microscopically detectable precipitate. These hamster cells readily facilitated the metabolism of 1,2-benzanthracene to a detectable mutagen and were especially competent in the activation of the two aromatic amines. Thus, cultured hamster hepatocytes can activate a variety of chemical carcinogens including polycyclic hydrocarbons to mutagens in a whole cell-mediated in vitro assay using L5178Y/TK^+/? cells as the target organism.     

Mutagenic evaluation of carcinogens and non-carcinogens in the L5178Y/TK assay utilizing postmitochondrial fractions (S9) from normal rat liver

As part of a continuing effort to investigate various metabolic activation procedures in the L5178Y TK^+/− → TK^−/− mutation assay, a series of 18 chemicals including both carcinogens and non-carcinogens selected from 7 chemical classes were tested in the presence of 5% (v/v) 9000 × g postmitochondrial supernatant fraction (S9) prepared from the livers of untreated Sprague-Dawley rats. Excepting ethyl carbamate and thioacetamide, 8 of 10 carcinogens tested produced mutant counts significantly greater than controls when the results were analyzed by a 2-sample log_et-test. Of 8 non-carcinogens assayed, treatment with p-aminophenol and chloroacetic acid yielded mutant counts significantly greater than control levels in at least one test with S9 while styrene oxide, an Ames positive mutagen, produced mutagenic activity in the absence of S9. These results indicate the L5178Y TK^+/− → TK^−/− mutation assay coupled with 5% (v/v) liver homogenate from normal rodent liver and applied to 18 chemicals correctly distinguished the majority of carcinogens from structurally related non-carcinogens on the basis of chemically-induced gene mutations.     

A microtiter plate assay for the selection of 6-thioguanine-resistant mutants in Chinese hamster V79 cells in the presence of phorbol-12-myristate-13-acetate

6-Thioguanine-resistant mutants can be efficiently recovered from Chinese hamster V79 cells incubated at high cell densities in microtiter plates (10³ – 10⁴ cells/0.2 ml growth medium/0.4 cm²) when selected with 30 μM 6-thioguanine and 0.1 μg/ml phorbol-12-myristate-13-acetate, an inhibitor of metabolic cooperation among V79 cells. Mutant frequencies in the microtiter plates were calculated from a direct count of mutant colonies. After treatment of the V79 cells with the carcinogen benzo[a]pyrene in a fibroblast-mediated assay, the mutation frequencies determined with the microtiter assay system were quantitatively similar to those obtained with a conventional procedure in which selection with 6-thioguanine was performed in petri dishes. The mutagenic activities of 3 polycyclic aromatic hydrocarbons (activated in the cell-mediated assay) were assessed with the microtiter plate selection procedure. The active carcinogen benzo[a]pyrene at 1 μg/ml yielded about 100 mutants per 10⁵ colony-forming cells. The same dose of a less active carcinogen, cyclopenta-[c,d]pyrene, yielded about 20 mutants per 10⁵ colony-forming cells, and benz[a]anthracene, not an active carcinogen, was inactive as a mutagen at all doses tested. Because of the small requirements for growth medium and tissue culture vessels compared with other assays, this microtiter plate assay can serve as an inexpensive system for detecting the mutagenic activity of environmental chemicals in mammalian cells.     

The effect of liver postmitochondrial fraction concentration from Aroclor 1254-treated rats on promutagen activation in L5178Y cells

L5178Y/TK^+/− cells were treated with either N-acetyl-2-aminofluorene (AAF), 2-aminoanthracene (2A), benzanthracene (BA), benzo[a]pyrene (BP), 3-methylcholanthrene (3MCA), or dimethylnitrosamine (DMN) in the presence of varied concentrations (2.5–10% v/v) of liver S9 (9000 × g) postmitochondrial fraction from Aroclor 1254-dosed male CD rats. Consistent S9 concentration-dependent decreases in trifluorothymidine-resistant (TFT^r) mutant induction were noted following 3 h exposure of L5178Y/TK^+/− cells to either 2.5 μg/ml BP, 50 or 67 μg/ml AAF, 0.96 μg/ml 2A, 7.35 μg/ml BA, or 5.4 μg/ml 3MCA. The exception was DMN which yielded a moderate S9 concentration-dependent increase in TFT^r mutant induction when L5178Y/TK^+/− cells were treated for 3 h with 74.1 μg/ml DMN. Depending upon the promutagen being tested, these results suggest two different metabolic events: (1) activation via non-induced enzymes or other factors whose activities are totally S9 volume-dependent (DMN), and (2) deactivation via induced detoxification pathways, a sequence favored by increasing S9 concentration (all others). Utilization of a single “standard” (10%) concentration of liver S9 from Aroclor 1254-treated rats did not appear to provide optimal metabolic activation for 5 of these 6 promutagens.     

Mutants with continuous microcycle conidiation in the filamentous fungus Fusarium solani f. sp. pisi

Summary A method of repeated mutation induction and subsequent selection was used to obtain mutants with microcycle conidiation, mc, in Fusarium solani f. sp. pisi. Wild-type, wt, Fusarium has a filamentous (hyphal) growth in a three-stage, conidium—hypha—conidiophore (phialid) vegetative life cycle. The first major mutation induced (by means of the mutagen MNNG) changed the wild-type strain 1–3^- from producing both macro- and microconidia to the mutant strain 19-P2^-, producing only microconidia but preserving the three-stage life cycle. When strain 19-P2^- was exposed to a similar mutagenic treatment mutants without the hyphal stage were obtained. In these mutants the microconidia germinate to form phialids directly, which in turn produce new conidia, thus accomplishing the life cycle as a two-stage, conidium-conidiophore, microcycle. The mc mutants were stabilized after reisolation through back-crossing to the wt of complementary mating type. Mc growth of the mutants is continuous under stable conditions, proceeding uninterrupted to the stationary stage in liquid as well as on solid medium. Mc colonies on solid medium are small and soft, well suited to replication by means of velvet or paper. Large numbers of mature, synchronized conidia are easily obtained from stationary-stage submerged cultures. Sequential mutations are of interest for studies of morphogenetic evolution. The ahyphal mutants make this group of eukaryotes well suited for genetic engineering and biotechnology.     

An in situ protocol for measuring the expression of chemically-induced mutations in mammalian cells

The generation of expression curves and the evaluation of mutagenic responses of mammalian cells using standard mutagenesis assays can be inaccurate because mutant and wild-type cells are usually mixed during the expression phase. If some mutant progenitors or mutants grow more slowly than the wild-type cells during the expression period, there will be a decrease in the mutant to wild-type ratio with time and the mutant fraction will not accurately represent the number of mutational events that occurred. The mutant fraction may also inaccurately assess the number of mutations if these mutations are expressed over a number of generations during the time before selection. We previously showed that recovery of L5178Y mouse cell mutants is not complete when mutations are allowed to express in suspension because slowly growing mutants and/or mutant progenitors are diluted out during this time (Rudd et al., 1990). In order to more accurately quantitate the mutagenic response of the cells, we developed an in situ procedure which segregates and immobilizes cells during expression. Because of this immobilization, slowly growing mutant progenitors and mutants expressed at different times will have an equal probability of being scored as mutants. Thus, one mutation leads to one mutant colony and the measurement of the mutagenic response of the cells to the chemical accurately reflects the mutational events that occurred. We plated L5178Y tk^+/− mouse cells in semisolid medium immediately after treatment. As the cells grew and formed microcolonies, the selective agent TFT was added as an overlay at specified times, permitting only TFT^r cells to survive. In this procedure, each mutation was captured as an individual colony; consequently, the measured mutation fraction accurately reflected the mutational events that occurred at the selected locus. In addition, the induced mutant colonies arising in the agar are the result of independent mutational events. We previously described the in situ protocol for L5178Y cells and showed that the spontaneous mutation rate measured was 50-fold greater than when the cells expressed the phenotype in suspension (Rudd et al., 1990). From this we concluded that the slow growth phenotype was expressed before TFT resistance. In the present paper, we evaluate the effect of chemical treatment on the mutation fraction as a function of the time to TFT addition. Using the in situ protocol, we generated expression curves for three nucleotide analogs, 5-azacytidine, TFT and AraC. The numbers of TFT^r colonies produced at various times after treatment indicated that chemically-treated cultures had higher mutation fractions than the solvent controls. The maximal differential increase in mutation rate occured between 30 and 60 h for 5-azacytidine and between 20 and 40 h for TFT and AraC. Our results document the feasibility of quantitating induced mutation fractions using the in situ protocol, confirm the mutagenicity of AraC and 5azacytidine and demonstrate the mutagenic activity of TFT at the tk locus. In addition to recovering mutants more accurately than the suspension protocol, the in situ protocol has the advantage of being experimentally less labor and time intensive. Therefore, we believe that this method should be considered for evaluation as an assay to measure the potential mutagenic effects of chemicals in mammalian cells in vitro.     

Some effects of bacteria populations on quantitation of ames salmonella-histidine reversion mutagenesis assays

We show from simple theory that the number of mutants observed in the Ames assay should be approximately proportional to αCn_oP where α is the mutation rate per concentration of mutagen, C; n₀ is the initial inoculum; and P is the average number of bacteria per colony in the background lawn. We tested this theory by carrying out Ames assay for the directly acting mutagen 2-nitrofluorene on TA98 as a function of initial inoculum over the range 10⁴–10⁸ bacteria/plate. P was assumed to be proportional to the average volume of the background colonies and was estimated from 100X photomicrographs of the background lawn. The initial inoculum was determined by counting background colonies in the photomicrographs, by dilution-plating, and by electronic particle counting. We found that the number of mutants depended weakly on n₀, but the dependence could be quantitatively accounted for by the simple theory. These data and the theory explain at least some of the reported variations in quantitation of the Ames assay; show that the slopes of dose— response curves depend on n₀; explain the range observed for spontaneous revertants; suggest some limitations to quantifying the Ames assay and, within these limits, suggest a method for normalizing independently obtained Ames assay data.     

Analysis of Cyanothece sp. BH68K mutants defective in aerobic nitrogen fixation

The unicellular cyanobacterium, Cyanothece sp. BH68K, is capable of performing both oxygen-sensitive nitrogen fixation and oxygenic photosynthesis within a single cell. To understand the oxygen protection mechanisms of nitrogenase, mutants defective in nitrogen fixation (Nif^-) were isolated by use of diethyl sulfate as a mutagen. Out of 24 mutants screened, 6 mutants could not express nitrogenase activity under aerobic conditions, but expressed activity under anaerobic conditions (Fox^-); 4 mutants showed no activity under both aerobic and anaerobic conditions (Fix^-); and the remaining mutants were impaired in both aerobic and anaerobic nitrogenase activity (Imp). Respiratory oxygen consumption and photosynthetic oxygen evolution were analyzed in the wild-type and in two Fox^- mutants. In the wild-type the appearance of high aerobic nitrogenase activity was correlated with an increase in dark respiration, whereas no such increase was seen in the Fox^- mutants. We propose that in Fox^- mutants, respiratory oxygen consumption plays an important role in maintaining aerobic nitrogenase activity.     

Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells II. Test validation and interpretation

The L5178Y Mouse Lymphoma TK assay was studied extensively to determine if this mammalian cell assay for gene mutations at the thymidine kinase (TK) locus could provide valid, interpretable determinations of mutagenic potential, and whether this information is of value in the safety evaluation of chemicals. We first determined that test-derived TFT^R mutants were phenotypically stable, possessing little or no thymidine kinase activity as measured by labeled thymidine uptake, but demonstrating 100% cross resistance to bromodeoxyuridine. Common solvent vehicles such as acetone, dimethylsulfoxide and ethanol were shown to produce little cytotoxicity and no mutagenic activity when present at 1% levels. Out of a total of 10 noncarcinogens tested, all were negative when results were analyzed by a 2-sample log_et test on control and treated mutant count means. Of the 13 putative animal carcinogens tested, 10 were positive, 2 were negative (auramine O and sodium phenobarbital), and 1 showed sporadic activity (hydrazine sulfate) in the TK assay on the basis of test-derived t statistics. 2 compounds, 1,2-epoxybutane and ICR 191, which have been described as Ames positive non-carcinogens, were also positive in the TK assay. Although this sampling of a total of 29 compounds is insufficient for precise estimations of expected false-positive or false-negative frequencies, these data indicate the TK assay can be expected to detect a majority of carcinogens as mutagens including some missed by more established point-mutation assays.     

An evaluation of the L5178Y TK+/− mouse lymophoma forward mutation assay using 42 chemicals

The L5178YTK^+/? mouse lymphoma assay (MLA) has been utilized in several laboratories as a short-term test for chemical-induced forward mutation in cultured mammalian cells. In order to evaluate several technical modifications to the MLA, 42 chemicals representing 9 chemical classes were tested and the results were compared with those published elsewhere as well as with findings in a genetic toxicology test battery currently used in this laboratory. A positive response for the induction of TK^+/? mutants was obtained for 26 chemicals. With the exception of p-aminophenol, all of these compounds were recognized mutagens or carcinogens and were represented of direct-acting and activation-dependent genotoxins. 16 compounds did not induce IK^?/? mutanants and among these were 5 compounds that were considered to be mutagens or carcinogens. A comparison of the results of this study with those published elsewhere revealed a strong agreement among findings for this test irrespective of minor technical variations. It was concluded that th MLA is a useful system for identifying chemical mutagens in mammalian cells and can serve as a valuabel component in a genetic toxicology test battery.     

Evidence for intrachromosomal gene conversion in cultured mouse cells

In this report we present an experimental scheme that facilitates the study of homologous recombination between closely linked genes in cultured mammalian cells. Two different Xho I linker insertion mutants of the herpes simplex virus type 1 thymidine kinase (HTK) gene were introduced into mouse LTK^? cells as direct repeats on a plasmid carrying a dominant selectable marker. Following stabilization of these sequences in the recipient cell, selection for TK⁺ was applied to detect recombinational events between different TK^? genes. TK⁺ segregants were observed at a frequency of 10^?4–10^?5 in lines harboring both mutant genes. Control lines carrying only one type of mutant HTK gene yielded TK⁺ cells at frequencies of 10^?7 or less. Physical analysis of the TK⁺ segregants has revealed the presence of an apparently normal HTK gene that is resistant to Xho I endonuclease digestion in each TK⁺ line examined. Analyses of the TK gene pairs before and after recombination suggest that at least 50% of the recombinants are the result of nonreciprocal exchanges of genetic information, or gene conversion events.     

ACQUISITION OF CHICK CYTOSOL THYMIDINE KINASE ACTIVITY BY THYMIDINE KINASE-DEFICIENT MOUSE FIBROBLAST CELLS AFTER FUSION WITH CHICK ERYTHROCYTES

Chick-mouse heterokaryons were obtained by UV-Sendai virus-induced fusion of chick erythrocytes with thymidine (dT) kinase-deficient mouse fibroblast [LM(TK^-)] cells. Autoradiographic studies demonstrated that 1 day after fusion, [³H]dT was incorporated into both red blood cell and LM(TK^-) nuclei of 23% of the heterokaryons. Self-fused LM(TK^-) cells failed to incorporate [³H]dT into nuclear DNA. 15 clonal lines of chick-mouse somatic cell hybrids [LM(TK^-)/CRB] were isolated from the heterokaryons by cultivating them in selective hypoxanthine-aminopterin-thymidine-glycine medium. LM(TK^-) and chick erythrocytes exhibited little, if any, cytosol dT kinase activity. In contrast, all 15 LM(TK^-)/CRB lines contained levels of cytosol dT kinase activity comparable to that found in chick embryo cells. Disk polyacrylamide gel electrophoresis and isoelectric focusing analyses demonstrated that the LM(TK^-)/CRB cells contained chick cytosol, but not mouse cytosol dT kinase. The LM(TK^-)/CRB cells also contained mouse mitochondrial, but not chick mitochondrial dT kinase. Hence, the clonal lines were somatic cell hybrids and not LM(TK^-) cell revertants. The experiments demonstrate that chick erythrocyte cytosol dT kinase can be activated in heterokaryons and in hybrid cells, most likely as a result of functions supplied by mouse fibroblast cells.     

Effect of DNA repair inhibitors on the induction and repair of bleomycin-induced chromosome damage.

The current status of the L5178Y/TK^+/-→TK^-/- mouse-lymphona mutagenicity assay is described. Dose-survival-mutagenic response data are shown for 43 chemicals. Mutagenicity and cytotoxicity in the presence or absence of non-induced and/or Aroclor-induced rat-liver S-9 are compared for most of these chemicals. 25 of these for which usable carcinogenicity data exist have been used to construct an approximately linear relationship between oncogenic potency in vivo and mutagenic potency in this system in vitro; linearity between these two endpoints extends over a greater than 100 000-fold range in potencies. Several carcinogens which are negative or difficult to detect in the standard Ames assay are mutagenic in this mammalian cell system. These include natulan, sodium saccharin (lot S-1022), p,p′-DDE (a metabolite of DDT), dimethylnitrosamine, diethylnitrosamine and diethylstilbestrol.     

The utilization of trifluorothymidine (TFT) to select for thymidine kinase-deficient (TK−/−) mutants from L5178Y/TK+/− mouse lymphoma cells

Trifluorothymidine (TFT), a thymidine analog, was analyzed for its ability to select for thymidine kinase-deficient (TK^−/−) mutants. In comparison with BUdR, the traditional selective agent for TK^−/− cells, it was determined that TFT at 1/50th the dose (1μg/ml vs. 50 μg/ml) is a more effective and versatile selective agent for TK^−/− mutants arising from the TK^+/−-3.7.2C heterozygote of L5178Y mouse lymphoma cells. Since TFT acts more rapidly than BUdR, it can be utilized in procedures (such as the analysis of the phenotypic lag) requiring the fast arrest of cell division. Reconstruction analyses of effective TK^−/− mutant recovery indicate that TFT can be used to recover mutants from significantly higher densities of TK^+/− cells than can BUdR. In addition, TK^−/− mutants can attain larger colony size in TFT than in BUdR where severe stunting of growth occurs at high TK^−/− cell densities. 190 of 194 isolated TFT-resistant large and small colony mutants (both spontaneous and induced)     

The Characterization of α-Glycerophosphate Dehydrogenase Mutants in DROSOPHILA MELANOGASTER

Thirty mutants of α-glycerophosphate dehydrogenase (αGPDH, EC 1.1.1.8) from Drosophila melanogaster were produced with the chemical mutagen ethyl methanesulfonate (EMS). These mutants and nine others previously obtained have been characterized with respect to level of enzymatic activity, viability, flight ability, and presence of cross-reacting material (CRM). The presence of αGPDH mRNA in several of the mutants has been tested by in vitro translation. There are strong correlations between the level of enzyme activity, viability and flight ability. Thirteen of the mutants are CRM^- by solution immunoprecipitation experiments, but of these, only three are CRM^- by a more sensitive ¹²⁵ I-protein A-based radioimmune gel assay. The viability of the three CRM^- mutants suggests that the absence of αGPDH protein is not a lethal condition. The immunoprecipitated protein of the low activity mutant, αGpdh^nGL3, has a smaller apparent molecular weight on polyacrylamide-SDS gels than does the protein from wild type. Criteria for the identification of nonsense mutations in Drosophila are discussed.     

Synergism of mutant frequencies in the mouse lymphoma cell mutagenicity assay by binary mixtures of methyl methanesulfonate and ethyl methanesulfonate

The effect of mixed mutagen exposures on the rate and type of induced mutants was studied in the L5178Y/TK+/-----TK-/- mouse lymphoma cell mutagenicity assay. In this assay, exposure to ethyl methanesulfonate (EMS) results in more mutants that form large colonies than small colonies. Exposure to methyl methanesulfonate (MMS) results in more mutants that form small colonies than large colonies. Other reports in the literature suggest that large colony TK-/- mutants appear to result from small-scale, perhaps single-gene mutations, and that small-colony TK-/- mutants appear to be associated with chromosomal mutations. Treating cells for 4 h with simple, 2-component mixtures containing 6.45 micrograms/ml MMS and either 261, 392, 560 or 712 micrograms/ml EMS resulted in synergism of mutants at each mixture level. The frequencies of total mutants were synergized 12, 20, 35 and 72%, respectively, in mixed exposures with graded doses of EMS, above the sums of the mixture components. Small colony mutants were synergized to a greater extent than large colony mutants. The frequencies of small colony mutants in mixed exposures were increased 31, 54, 73 and 123%, respectively, while the frequencies of large colony mutants were increased -7, -6, 11 and 39%. Statistical analyses provide strong evidence of synergism (within the limits of the assay) for total and small-colony mutants at all doses of EMS tested, and for large-colony mutants above 400 micrograms/ml EMS. Similar magnitudes of synergism resulted when other constant levels of MMS (4.30 or 8.60 micrograms/ml) were mixed with the same graded doses of EMS. The degree of synergism was dependent on EMS concentration but not on MMS concentration.     

The induction of thymidine- and IUdR-resistant variants in P388 mouse lymphoma cells by x-rays, UV and mono- and bi-functional alkylating agents

Dose-response curves for “mutation” to resistance to 5-iodo-2-deoxyuridine (IUdR) and excess thymidine (TdR) in P388 mouse lymphoma cells have been established after exposure of these cells to six chemical mutagens, UV |and| ionising radiations. The dose-response curves for all mutagens in both selective system show considerable similarities when induced mutation frequencies are plotted against survival. Expression time for both types of variants, IUdR^r and TdR^r, are similar, i.e. maximum frequencies are reached by 48 h and there is no fall in variant frequency at late expression times up to 144 h. Over the range of survival levels studied there appears to be little or no dependence of expression time on dose of mutagen. Some loss of mutants after high doses (i.e. at low survival levels) was observed due to the fact that a significant proportion of both TdR^r and IUdR^r clones were more sensitive to the mutagens than the wild-type population. The similarities in induced dose-response curves for different mutagens suggest that the mutants have a common origin, probably an error in repair, but it seems unlikely that errors in “cut and patch” repair are responsible. A comparison of spontaneous frequencies of IUdR^r and TdR^r variants suggests that IUdR is mutagenic in P388 cells.