Phenotypic expression time of mutagen-induced 6-thioguanine resistance in Chinese hamster ovary cells (CHO/HGPRT system).

Mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells (referred to as the CHO/HGPRT system) can be quantitated by selection for the phenotype of resistance to 6-thioguanine (TG) under stringently defined conditions. The phenotypic expression time, that is, the time interval after mutagen treatment which is necessary befor all mutant cells are able to express the TG-resistant phenotype, has been found to be 7–9 days in this CHO/HGPRT system when the cells are subcultured every 48 h. Subculture in medium with or without hypoxanthine (HX) utilizing trypsin, ethylenediaminetetraacetic acid (EDTA), or ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) for cell removal yields identical results. When subculture at intervals greater than 48 h is employed, a slight lengthening of the expression time is observed. An alternative method to regular subculture has also been achieved by maintaining the cells in a viable, non-dividing state in serum-free medium. This procedure yields a similar time course of phenotypic expression and thus shows that continued cell division is not essential to this expression process. In addition, this observation offers methodology which can significantly reduce the investment of time and money for mutation induction determinations in this mammalian cell gene mutation assay.     

Mutagenicity and cytotoxicity of congeners of two classes of nitroso compounds in Chinese hamster ovary cells

The induction of mutation by certain nitrosamidines and nitrosamides has been quantitated utilizing the hypoxanthine--guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary cells. Dose--response relationships for cytotoxicity and mutagenicity are presented for N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), N-butyl-N-nitrosourea (BNU), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). Based on the concentration of each agent required to kill 90% of the cells, the following order of cytotoxicity was observed: MNNG greater than ENNG greater than MNU greater than ENU greater than BNU. This is the same order of potency as observed for mutation induction per unit concentration of mutagen.     

An effect of cell-cycle position on ultraviolet-light-induced mutagenesis in Chinese hamster ovary cells

Using synchronous populations obtained by selectively detaching mitotic cells from cultures grown in monolayer, we demonstrate here that Chinese hamster ovary (CHO) cells exhibit a differential sensitivity to mutation induction by UV as a function of position in the cell cycle. When mutation induction to 6-thioguanine (TG) resistance is monitored, several maxima and minima are displayed during cell-cycle traverse, with a major maximum occurring in early S phase. Although cells in S phase are more sensitive to UV-mediated cell lethality than those in G₁ or G₂/M phases, there is not a strict correlation with induced mutation frequency. Fluence-response curves obtained at several times during the cell cycle yield D_q values approximating 6 J/m². The primary survival characteristic which varies with cell cycle position is D₀, ranging from 2.5 J/m² at 6 h after mitotic selection to 5.5 J/m² at 11 h afterward. Based on studies with asynchronous, logarithmically growing populations, as well as those mitotically selected to be synchronous, the optimum phenotypic expression time for induced TG resistance is 7–9 days and is essentially independent of both UV fluence and position in the cell cycle. All isolated mutants have altered hypozanthine—guanine phosphoribosyl transferase (HGPRT) activity, and no difference in the residual level of activity was detected among isolated clones receiving UV radiation during G₁, S, or late S/G₂ phases of the cell cycle. Changes in cellular morphology during cell-cycle traverse do not contribute to the differential susceptibility to UV-induced mutagenesis.     

Quantification and analysis of reverse mutations at the hgprt locus in Chinese hamster ovary cells

We describe an assay for the quantification of reverse mutations at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus in Chinese hamster ovary cells utilizing the selective agent L-azaserine (AS). Conditions are defined in terms of optimal AS concentration, cell density, and phenotypic expression time. After treatment, replicate cultures of 10⁶ cells are allowed a 48-h phenotypic expression time in 100-mm plates. AS (10 μM) is then added directly to the growing culture and AS-resistant (AS^r) cells form visible colonies. This assay is used to quantify ICR-191-, ICR-170-, and N-ethyl-N-nitrosourea-induced reversion of independently isolated HGPRT^? clones. The AS^r phenotype is characterized both physiologically and biochemically. All AS^r clones isolated are stably resistant to AS and aminopterin but sensitive to 6-thioguanine. They also have re-expressed HGPRT enzyme. In addition, several revertants are shown to contain altered HGPRT. The data provide further evidence that ICR-191 and ICR-170 cause structural gene mutations in mammalian cells and also suggest that ICR-191, ICR-170, and N-ethyl-N-nitrosourea induce similar types of mutations in Chinese hamster ovary cells.     

Further evidence for the genetic origin of mutations in mammalian somatic cells: the effects of ploidy level and selection stringency on dose-dependent chemical mutagenesis to purine analogue resistance in Chinese hamster ovary cells.

Whether resistance to purine analogues 8-azaguanine (AG) and 6-thioguanine (TG) in mammalian cells is due to gene mutation or to epigenetic changes was investigated by an ethyl methanesulfonate (EMS) dose-dependent induced “resistance” to these analogues in two near-diploid (2N) and one tetraploid (4N) Chinese hamster ovary (CHO) cells. EMS produced higher cell killing in 2N than in 4N cells. In the 2N cells, EMS-induced mutations to TG (1.7 μg/ml) resistance increased approximately as a linear function of the dose from 0–400 μg/ml. However, EMS was ineffective in inducing such mutation in the 4N cells. These observations are consistent with the notion that the induced TG resistance arose as a result of mutation at the gene or chromosome level. In each cell type, both the “observed” spontaneous and the EMS-induced frequency to purine analogue resistance decreased with increasing concentration of purine analogues. However, among the “resistant” clones a high proportion of those selected at 1.2 and 3.0 μg/ml of AG, a small portion selected at 7.5 μg/ml of AG, and virtually none at 1.7 and 6.0 μg/ml of TG are capable of growth in medium containing aminopterin (10 μM). This suggests that, under less stringent selective conditions, some resistant variants were being selected through mechanisms not yet defined.     

Phenotypic lag and mutation to 6-thioguanine resistance in diploid human lymphoblasts.

Mutants of a diploid human lymphoblast line resistant to 6-thioguanine (6TG) appear 6--16 generations after treatment with any of a diverse group of mutagents: methylnitrosourea (MNU), methylnitrosoguanidine (MNNG), ICR-191, 5-bromodeoxyuridine (BUdR). A hypothesis is advanced that expression of the 6-thioguanine-resistant state may require the removal of essentially all pre-existing hypoxanthine--guanine phosphoribosyl transferase (HGPRT) molecules via division, dilution, and protein turnover. Design of protocols for quantitative mutation assays requires attention to this phenomenon.     

In vitro metabolic activation of ethidium bromide and other phenanthridinium compounds: mutagenic activity in Salmonella typhimurium.

The induction of mutation by a variety of mutagens has been measured utilizing the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells (CHO/HGPRT) system). These mutagens include physical agents such as UV light and X-rays, and chemicals such as alkylating agents, ICR-191, and metallic compounds. This system can also be modified for study of the mutagenicity of promutagens such as dimethylnitrosamine (DMN) which require biotransformation for mutagenic action, either through the addition of a rat liver microsomal activation preparation or through a host-mediated activation step using Balb/c athymic mice.     

The relative effectiveness of 6-thioguanine and 8-azaguanine in selecting resistant mutants from two V79 Chinese hamster cells in vitro

The frequency of surviving colonies in two V79 cell lines exposed to either 6-thioguanine or 8-azaguanine was dependent on initial plating density. Different degrees of metabolic-co-operation were found to occur in the two cell lines and the loss of both spontaneous and added mutants occurred at a lower cell density when 6TG was used for selection than when 8 AZ was used in both cell lines. Both analogues were degraded on incubation in medium plus serum in the absence of added cells. Variation in serum batch had little effect on the rate of degradation or on the frequency of colonies recovered after treatment of V79 cell lines with 8AZ. The reasons for preferring 8AZ to 6TG as a selective agent are discussed.     

Conditions necessary for quantifying ethyl methanesulfonate-induced mutations to purine-analogue resistance in Chinese hamster V79 cells.

We have investigated conditions necessary to quantify the relationship between exposure to a mutagen, ethyl methanesulfonate (EMS), and the frequency of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in V79 cells. Maximal expression of potential mutants has been achieved by either subculturing at fewer than 5 X 10(5) cells/100-mm dish at 2-day intervals or by daily feeding of cultures. An expression period of 5 days (measure from 1 day after the initiation of treatment with the chemical mutagen) should be allowed, since at least 4 days of expression is required to reach to steady maximum of mutation frequency. It appears that there is no concentration dependence of expression time necessary to reach a plateau of mutation frequency with increasing concentrations of EMS up to 1.6 mg/ml. About 1.25 X 10(5) cells/100-mm dish or fewer should be plated for selection to avoid the loss of mutants which occurs at 1.5 X 10(5) cells/dish, presumably through cross-feeding (metabolic cooperation). The use of 6-thioguanine in hypoxanthine-free medium (supplemented with dialyzed fetal calf serum) appears to be a very stringent condition for selection. Mutation induction by EMS as a function of EMS exposure (EMS concentration X treatment time) increases linearly with concentration up to 12 h. For these treatment periods, the observed mutation frequencies for EMS are directly proportional to mutagen exposure regardless of the duration of the treatment.     

Comparative mutagenicity of alkylsulfate and alkanesulfonate derivatives in Chinese hamster ovary cells.

Mutation induction and cell killing produced by selected alkylsulfates and alkanesulfonates have been quantitated using the Chinese hamster ovary/hypoxanthine--guanine phosphoribosyl transferase (CHO/HGPRT) system. Dose--response relationships of cytotoxicity and mutagenicity are presented for two alkylsulfates [dimethylsulfate (DMS), diethylsulfate (DES)] and three alkyl alkanesulfonates [methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), and isopropyl methanesulfonate (iPMS)]. Under the experimental conditions employed, cytotoxicity decreased with the size of the alkyl group. DMS was more toxic than DES, and MMS was more toxic than EMS and iPMS. All agents produced linear dose--response of mutation induction: DMS was more mutagenic than DES, and MMS was more mutagenic than EMS and iPMS based on mutants induced per unit mutagen concentration. However, the following relative mutagenic potency was observed when comparisons were made at 10% survival: DES greater than DMS; EMS greater than MMS greater than iPMS.     

Induction and expression of mutations in mammalian cells in the absence of DNA synthesis and cell division

The induction of mutations by the alkylating agent ethyl methanesulfonate (EMS) was determined with Chinese hamster ovary cells maintained in serum-free medium to arrest DNA synthesis and cell division. The arrested cultures were treated with EMS and maintained in serum-free medium for various time intervals post-treatment before serum containing medium was added to initiate DNA synthesis and cell division. The concentration-dependent increase in 6-thioguanine-resistant mutants in the arrested cultures was similar to that found with exponentially dividing cultures when serum was added to the arrested cultures immediately after the EMS treatment; the time course of phenotypic expression was also similar with both cultures. In addition, maintenance of the arrested cultures in serum-free medium for up to 18 days post-treatment resulted in no change in the mutant frequency. This suggests that the mutagenic damage is not removed in these arrested cultures. Furthermore, maintenance of the arrested state for increasing time intervals before serum addition results in decreases in the time necessary for maximum phenotypic expression. Cultures maintained in serum-free medium for 16 days after mutation treatment show complete expression of the mutations with no need for subculture. This last result suggests that the mutagenic damage induced by EMS in Chinese hamster ovary cells is not removed and that this damage results in both the induction and expression of mutation in the absence of DNA replication.     

Induction of 6-thioguanine resistance in human cells treated with N-acetoxy-2-acetylaminofluorene

Induction of 6-thioguanine resistance was studied in human cells treated with the direct-acting chemical carcinogen N-acetoxy-2-acetylaminofluorene (NA-AAF). At low concentrations (2.5–7.5 μM) induction of resistant clones was linear and followed one-hit kinetics, while at 10 μM the yield of resistant clones was higher and appeared to result from the combination of one-hit and two-hit kinetics. A study of about 50 resistant clones revealed that most had reduced levels of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity (25–85% of controls) and were able to use exogenous hypoxanthine for growth (“Type II mutants,” deMars, 1974); a few had very low HGPRT activity (1–8% of controls) and were unable to use exogenous hypoxanthine (“Type I mutants”). Use of [9¹⁴-C]NA-AAF allowed us to examine the frequency of induction of thioguanine resistance as a function of binding to DNA (μmole AAF/mole DNA-P). Calculations from these data suggest that most “hits” on the HGPRT locus do not result in detectable mutations: At three different levels of binding and induced mutation frequency, the yield was 2.5–3 detectable mutants/10 000 molecules of acetylaminofluorene bound to the HGPRT locus. These data suggest that most bound acetylaminofluorene molecules either produce no change in the primary sequence of DNA (possibly as a result of repair or correct “read through” by the DNA polymerase) or result in changes which are phenotypically undetectable.     

Correlation between specific DNA-methylation products and mutation induction at the HGPRT locus in Chinese hamster ovary cells

Suspension cultures of Chinese hamster ovary (CHO) cells were exposed to methyl methanesulfonate (MMS) or methylnitrosourea (MNU) and assayed for mutation induction (6-thioguanine resistance) and for specific DNA adducts. DNA methylation at the 1-, 3- and 7-positions of adenine, the 3-, O6- and 7-positions of guanine, and phosphate was detected in cultures exposed to MMS, while MNU produced 3- and 7-methyladenine, 3-methylcytosine, 3-, O6- and 7-methylguanine, O4-methylthymidine and methylated phosphodiesters. When mutations induced by MMS and MNU were compared by linear correlation analysis with levels of each of these adducts, only O6-methylguanine displayed a strong correlation with mutations (r = 0.879, p less than 0.001). The relationship between O6-methylguanine and induced mutations in CHO cells is similar to that previously reported in CHO cells for O6-ethylguanine and mutations (Heflich et al., 1982) and indicates that alkylation-induced mutations at the HGPRT locus in CHO cells are primarily associated with O6-alkylguanine formation.     

Mutagenicity of an antitumor protein, neocarzinostatin, in Escherichia coli.

An assay is described for the measurement of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells utilizing resistance to 6-thioguanine (TG). Optimal selection conditions are defined for such parameters as phenotypic expression time prior to selection, and TG concentration and cell density which permits maximum mutant recovery. The nature of the TG-resistant mutants is characterized by several physiological and biochemical methods. The data demonstrate that more than 98% of the mutant clones isolated by this selection procedure contain altered HGPRTase activity. The CHO/HGPRT system thus shows the specificity necessary for a specific gene locus mutational assay.     

Induction of 6-thioguanine resistance in human cells treated with N-acetoxy-2-acetylaminofluorene

Induction of 6-thioguanine resistance was studied in human cells treated with the direct-acting chemical carcinogen N-acetoxy-2-acetylaminofluorene (NA-AAF). At low concentrations (2.5–7.5 μM) induction of resistant clones was linear and followed one-hit kinetics, while at 10 μM the yield of resistant clones was higher and appeared to result from the combination of one-hit and two-hit kinetics. A study of about 50 resistant clones revealed that most had reduced levels of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity (25–85% of controls) and were able to use exogenous hypoxanthine for growth (“Type II mutants,” deMars, 1974); a few had very low HGPRT activity (1–8% of controls) and were unable to use exogenous hypoxanthine (“Type I mutants”). Use of [9-¹⁴C]NA-AAF allowed us to examine the frequency of induction of thioguanine resistance as a function of binding to DNA (μmole AAF/mole DNA-P). Calculations from these data suggest that most “hits” on the HGPRT locus do not result in detectable mutations: At three different levels of binding and induced mutation frequency, the yield was 2.5-3 detectable mutants/10 000 molecules of acetylaminofluorene bound to the HGPRT locus. These data suggest that most bound acetylaminofluorene molecules either produce no change in the primary sequence of DNA (possibly as a result of repair or correct “read through” by the DNA polymerase) or result in changes which are phenotypically undetectable.     

Cycloheximide resistance in Chinese hamster cells. II. Induction of Chm resistance in Chinese hamster cells by N-nitrosomethylurea.

Mutant Chinese hamster ovarian (CHO) cells with a resistance to 7-10(-7) and 8-10(-7) M cycloheximide (CHM) were induced at mutation rates of 1.9-5.2-10(-3) and 1.6-1.8-10(-3) respectively after treatment with N-nitrosomethylurea (NMU) at 100 mug/ml. The induced mutation rates differed by two orders of magnitude from the spontaneous rate of mutation to CHM resistance.     

Dilution of hypoxanthine-guanine phosphoribosyl transferase and other factors affecting the frequency of 6-thioguanine resistance in Chinese hamster lung cells.

Factors influencing the frequency of thioguanine resistant mutations were examined in Chinese hamster lung cells damaged with a carcinogen, N-acetoxy-2-acetyl aminofluorene. Factors such as inoculum density, expression time, and concentration of selective agent were found to have a profound effect on the mutation frequency.Over a range of doses, a longer expression time is required for mutant cells from a more damaged population to reach their maximum frequency. In order to investigate the elements involved in this phenomenon, the increment in the plating efficiency of treated cells as a function of expression time, spontaneous mutation rate per cell per generation, viability of mutant as well as wild type cells, and half life of HGPRTase were evaluated.There was an observed relationship between induced mutation frequency and plating efficiency of treated cells. When treated cells had recovered from effects of the treatment and arrived at the normal level of plating efficiency, they also yielded the maximum frequency of mutations.The estimated mutation rate was 5.5 × 10^?8 per cell per generation. This number is too small to account for the increment in mutation frequency with the increase in the expression time. The mutation frequency of spontaneous origin was 4 × 10^?6 and that of induction of 10^?5 M NA-AAF was 10^?4. Lower growth rates of mutant cells cannot explain this increase in the number of mutants recovered, either.Continuous diminution in the level of HGPRTase, at 35% daily, interpreted as an important factor responsible for the recovery of mutation frequency during expression time, was observed in non-dividing cells. None of a large number of mutants sampled from those isolated had HGRPT activity. This indicates that they are true mutants and are not a result of phenocopy. Only cells completely deficient in HGPRT activity are recovered in TG selection medium. It is suggested, therefore, that this cell line is suitable for mutagenicity testing in the induction of mutation at the HGPRT locus.     

Relationship between DNA alkylation and specific-locus mutation induction by N-methyl- and N-ethyl-N-nitrosourea in cultured Chinese hamster ovary cells (CHO/HGPRT system)

Chinese hamster ovary (CHO) cells in culture were utilized to determine the cytotoxicity, specific-locus mutation induction, and DNA alkylation which result from treatment of the cells with a range of concentrations of N-methyl-N-nitrosourea (MNU) or N-ethyl-N-nitrosourea (ENU). With [3H]MNU over the concentration range 0.43--13.7 mM, methylation of DNA was found to increase linearly, with a mean value of 56.7 pmol residue per mumol nucleoside per mM. With [1-3H]ENU over the concentration range 1.7--26.8 mM, ethylation was linear, with a mean value of 3.8 pmol residue per mumol nucleotide per mM. Mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus was quantified by determination of the frequency of resistance to 6-thioguanine under stringently-defined selection conditions. The mutation frequency increased linearly with MNU or ENU concentration (0.01--2.0 mM); mean values were 2800 and 840 mutants per 10(6) clonable cells per mM, respectively. At equal levels of DNA alkylation, ENU was found to be approx. 4.5 times as mutagenic as MNU.     

Effect of calcium phosphate and alumina C γ gels on the mutagenicity and cytotoxicity of dimethylnitrosamine as studied in the CHO/HGPRT system

When CaCl₂ was added in increasing concentrations to a rat liver metabolic activation system (S9) buffered with sodium phosphate, the mutagenic activity and cytotoxicity of dimethylnitrosamine (DMN) in the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) system were greatly increased. This effect was not observed with an S9 mix buffered with N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES). The calcium phosphate gel precipitate of the S9 mix possessed approximately $\text{built1}\text{3}$ of the total activity of the mix, while the supernatant had only slight activity. However, when the calcium phosphate gel precipitate of a solution of S9 salts (without S9 protein) was added to the supernatant, the remaining $\text{2}\text{3}$ of the activity was recovered. Commercially obtained calcium phosphate, tricalcium phosphate, and alumina C γ gels could substitute for CaCl₂ in the S9 mix, but diethylaminoethyl cellulose (DEAE cellulose) could not. Alumina C γ gel can exert its effect in the absence of both CaCl₂ and phosphate in the S9 mix. Increasing the time of contact between the S9 protein and the S9 salts increased the efficacy with which the S9 mix activated DMN; this is indicative of an adsorptive process by calcium phosphate gel.     

Clones of Chinese hamster cells cultivated in vitro not permanently resistant to azaguanine

The frequency of clones not permanently resistant to azaguanine (AG) was measured in Chinese hamster ovary cells (CHO) grown in vitro by plating them in 7.5 μg/ml AG and isolating a number of clones in the course of 5 experiments. Such isolated clones were propagated to a point at which their resistance to both AG and the reverse selective medium, HAT, could be determined. Out of a total of 13 clones isolated, 4 of these could not be distinguished from the parent CHO line, either on the basis of their growth in a gradient of AG concentrations or the reverse selective HAT medium or on the basis of their mutation frequency to resistance to 30 μg/ml AG. All four of the apparent phenocopies were isolated from plates in which although lower numbers of cells were seeded, a higher frequency of clones able to grow in AG was yielded. This suggests that the higher “mutation” frequencies obtained at lower cell densities are due to the appearance of phenocopies which occur only under these conditions. It is concluded that under low plating density conditions, the lower levels of AG (7.5 μg/ml) are not satisfactory for mutagenesis and mutation rate studies.