Relative mutagenicity of antineoplastic drugs and other alkylating agents in V79 chinese hamster cells,independence of cytotoxic and mutagenic responses

The mutagenic and cytotoxic effects of 4 antineoplastic drugs, vinblastine, vincristine, adriamycin and nitrogen mustard and of several monofunctional alkylating agents have been assayed in V79 Chinese hamster cells. Vincristine, vinblastine and nitrogen mustard did not significantly increase the frequency of TG^RHGPRT^? mutants but were all highly cytotoxic. Adriamycin and the monofunctional alkylating agents were all significantly mutagenic even at the lowest doses tested (approx. 70 % survival level). Induced mutant frequency increased linearly with increasing dose whereas dose-response curves for cytotoxicity for these effective mutagens invariably showed a shoulder followed by an exponential decline. At equitoxic doses the relative mutagenic effectiveness was MNU ENU EMS MMS ? DMS. MNU was approx. 20 times more effective than MMS and DMS.Measurement of the total amount of alkylation and the relative amounts of reaction with individual DNA bases at approx. equitoxic doses of MNU and DMS indicated a significantly higher O⁶/N⁷ ratio after MNU (0.15) than after DMS (0.005). However, approx. equal numbers of mutants/10⁵ cells/μM O⁶-Meguanine were induced by these 2 agents. These results support previous conclusions, that mutagenic and cytotoxic responses are independent in V79 cells.     

Toxicity and mutagenicity of X-rays and [I]dUrd or [H]TdR incorporated in the DNA of human lymphoblast cells

We measured the toxicity and mutagenicity induced in human diploid lymphoblasts by various radiation doses of X-rays and two internal emitters. [¹²⁵I]iododeoxyuridine ([¹²⁵I]dUrd) and [³H]thymidine ([³H]TdR), incorporated into cellular DNA. [¹²⁵I]dUrd was more effective than [³H]TdR at killing cells and producing mutations to 6-thioguanine resistance (6TG^R). No ouabain-resistant mutants were induced by any of these agents. Expressing dose as total disintegrations per cell (dpc), the D₀ for cell killing for [¹²⁵I]dUrd was 28 dpc and for [³H]TdR was 385 dpc. The D₀ for X-rays was 48 rad at 37°C. The slopes of the mutation curves were approximately 75 × 10⁻⁸ 6TG^R mutants per cell per disintegration for [¹²⁵I]dUrd and 2 × 10⁻⁸ for [³H]TdR. X-Rays induced 8 × 10⁻⁸ 6TG^R mutants per cell per rad. Normalizing for survival, [¹²⁵I]dUrd remained much more mutagenic at low doses (high survival levels) than the other two agents. Treatment of the cells at either 37°C or while frozen at −70°C yielded no difference in cytotoxicity or mutation for [¹²⁵I]dUrd or [³H]TdR, whereas X-rays were 6 times less effective in killing cells at −70°C.Assuming that incorporation was random throughout the genome, the mutagenic efficiencies of the radionuclides could be calculated by dividing the mutation rate by the level of incorporation. If the effective target size of the 6TG^R locus is 1000–3000 base pairs, then the mutagenic efficiency of [¹²⁵I]dUrd is 1.0–3.0 and of [³H]TdR is 0.02–0.06 total genomic mutations per cell per disintegration. ¹²⁵I disintegrations are known to produce localized DNA double-strand breaks. If these breaks are potentially lethal lesions, they must be repaired, since the mean lethal dose (D₀) was 28 dpc. The observations that a single dpc has a high probability of producing a mutation (mutagenic efficiency 1.0–3.0) would suggest, however, that this repair is extremely error-prone. If the breaks need not be repaired to permit survival, then lethal lesions are a subset of or are completely different from mutagenic lesions.     

Inhibition of post-replication repair of alkylated DNA by caffeine in Chinese hamster cells but not HeLa cells

Caffeine has been found to potentiate the lethal effects of sulphur mustard (SM) and N-methyl-N-nitrosourea (MNU) in a line of Chinese hamster cells but not in a line of HeLa cells. The sensitization of SM-treated cells by caffeine was S phase specific, and persisted for up to 24 h after alkylation of asynchronous cell cultures. The sensitization of MNU-treated cells, however, was not S phase specific but persisted for up to 50 h after the initial alkylation. Possible explanations for this difference between these two types of alkylating agent were discussed. Previously, evidence was presented which suggested that the alkylation-induced delay in the time of the peak rate of DNA synthesis in Chinese hamster cells was associated with the operation of post-DNA replication repair mechanism in these cells. Caffeine has now been found to reverse this alkylation-induced delay of DNA synthesis in both SM- and MNU-alkylated Chinese hamster cells. It is therefore proposed that caffeine sensitizes alkylated cells by inhibition of a post-replication DNA repair mechanism. No support was obtained for the alternative possibility that caffeine inhibits alkylation-induced excision repair of damaged DNA. The role of DNA repair in the production of the lethal mutagenic and cytological effects of alkylating agents is discussed.     

Characteristics of spontaneous and induced thymidine and 5-iodo-2-deoxyuridine resistant clones of mouse lymphoma cells

Clones resistant to 5-iodo-2-deoxyuridine (IUdR) were isolated from P388 cells and cultured in the absence of selective medium. Thymidine kinase assays were performed on 8 clones which had arisen spontaneously and 19 isolated after exposure to X-rays or alkylating agents. All the clones tested showed significantly reduced thymidine kinase activity relative to wild-type cultures, but none showed zero levels. 14 of these clones were tested for thymidine (TdR) uptake and all showed a marked reduction in the rate of [³H]TdR incorporation into acid soluble fractions and into DNA. 7 IUdR-resistant (IUdR^r) clones were tested for revertibility as measured by growth of colonies in HAT medium. 5 of the 7 were found to revert at measurable rates either spontaneously or after a low dose of mutagen.Thymidine kinase activity was also measured in 8 thymidine resistant P388 clones (TdR^r). Initial rates of thymidine phosphorylation were not significantly altered in 5 of the 8 clones tested but significantly lower amounts of phosphorylated products were observed in 6 of the 8 clones. [³H]TdR uptake was reduced in 9 of 12 clones tested, and 2 of them showed no corresponding reduction in the thymidine kinase activity, suggesting the occurence of mutants with altered permeability for thymidine.IUdR resistant L5178Y clones could not be isolated. Thymidine resistant L5178Y clones were similar to TdR^r P388 clones, i.e. they showed changes in initial rates of thymidine kinase activity and reduced accumulation of phosphorylated products. Only one clone could be shown to be a membrane mutant. These results are discussed in relation to the genetic nature of the thymidine kinase locus in the two cell lines.     

Differential sensitivity of DNA replication and repair in permeable Escherichia coli exposed to various monofunctional methylating or ethylating agents.

Escherichia coli cells made permeable to deoxynucleoside triphosphates by brief treatment with toluene (permeablized) were used to measure the effect of the following chemical alkylating agents on either DNA replication or DNA repair synthesis: methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and N-ethyl-N′-nitro-N-nitrosoguanidine (ENNG). Replication of DNA in this pseudo-in vivo system was completely inhibited 10–15 min after exposure to MMS at concentrations of 5 mM or higher or to MNU or MNNG at concentrations of 1 mM or higher. The ethyl derivatives of the alkylating agents were less inhibitory than their corresponding methyl derivatives, and inhibition of DNA replication occurred in the following order: EMS < ENNG < ENU. Maximum inhibition of DNA replication by all of the alkylating agents tested except EMS occurred at a concentration of 20 mM or lower. The extent of replication in cells exposed to EMS continued to decrease with concentrations of EMS up to 100 mM (the highest concentration tested).The experiments in which the inhibition of DNA replication by MMS, MNU, or MNNG was measured were repeated under similar assay conditions except that a density label was included and the DNA was banded in CsCl gradients. The bulk of the newly synthesized DNA from the untreated cells was found to be of the replicative (semi-conservative) type. The amount of replicative DNA decreased with increasing concentration of methylating agent in a manner similar to that observed in the incorporation experiments.Polymerase I (Pol I)-directed DNA repair synthesis induced by X-irradiation of permeablized cells was assayed under conditions that blocked the activity of DNA polymerases II and III. Exposure of cells to MNNG or ENNG at a concentration of 20 mM resulted in reductions in Pol I activity of 40 and 30%, respectively, compared with untreated controls. ENU was slightly inhibitory to Pol I activity, while MMS, EMS, and MNU all caused some enhancement of Pol I activity.These data show that DNA replication in a pseudo-in vivo bacterial system is particularly sensitive to the actions of known chemical mutagens, whereas DNA repair carried out by the Pol I repair enzyme is much less sensitive and in some cases apparently unaffected by such treatment. Possible mechanisms for this differential effect on DNA metabolism and its correlation with current theories of chemically induced mutagenesis and carcinogenesis are discussed.     

Post-replication DNA repair in barley embryos treated with N-methyl-N-nitrosourea

In sterile cultures of free barley embryos, N-methyl-N-nitrosourea (MNU) caused a decrease in the size of both template [¹⁴C]-labeled DNA and of daughter [³H]DNA strands as determined in alkaline sucrose gradients, and inhibited the rate of [³H]thymidine incorporation. In addition, duplexes containing [³H]-daughter DNA analyzed in BND cellulose contained more single-stranded regions in MNU-treated embryos than in the corresponding control. Incubation of MNU-treated embryos in nutrient medium for up to 18 h after the [³H]-labeling permitted the recovery of small-sized daughter DNA to full-sized strands and led to the enhancement of double-strandedness of DNA duplexes containing [³H]-labeled strands. If [³H]-labeling had been carried out 8–10 h after the MNU treatment, the size of daughter DNA, the proportion of double-strandedness and the rate of thymidine uptake into DNA partially increased in comparison with rates observed when labeling had been done just after or 3 h after the MNU treatment, but these variables did not reach the values of the corresponding controls.     

Toxicity and mutagenicity of X-rays and [125I]dUrd or [3H]TdR incorporated in the DNA of human lymphoblast cells

We measured the toxicity and mutagenicity induced in human diploid lymphoblasts by various radiation doses of X-rays and two internal emitters. [¹²⁵I]iododeoxyuridine ([¹²⁵I]dUrd) and [³H]thymidine ([³H]TdR), incorporated into cellular DNA. [¹²⁵I]dUrd was more effective than [³H]TdR at killing cells and producing mutations to 6-thioguanine resistance (6TG^R). No ouabain-resistant mutants were induced by any of these agents. Expressing dose as total disintegrations per cell (dpc), the D₀ for cell killing for [¹²⁵I]dUrd was 28 dpc and for [³H]TdR was 385 dpc. The D₀ for X-rays was 48 rad at 37°C. The slopes of the mutation curves were approximately 75 × 10⁻⁸ 6TG^R mutants per cell per disintegration for [¹²⁵I]dUrd and 2 × 10⁻⁸ for [³H]TdR. X-Rays induced 8 × 10⁻⁸ 6TG^R mutants per cell per rad. Normalizing for survival, [¹²⁵I]dUrd remained much more mutagenic at low doses (high survival levels) than the other two agents. Treatment of the cells at either 37°C or while frozen at −70°C yielded no difference in cytotoxicity or mutation for [¹²⁵I]dUrd or [³H]TdR, whereas X-rays were 6 times less effective in killing cells at −70°C.

Assuming that incorporation was random throughout the genome, the mutagenic efficiencies of the radionuclides could be calculated by dividing the mutation rate by the level of incorporation. If the effective target size of the 6TG^R locus is 1000–3000 base pairs, then the mutagenic efficiency of [¹²⁵I]dUrd is 1.0–3.0 and of [³H]TdR is 0.02–0.06 total genomic mutations per cell per disintegration. ¹²⁵I disintegrations are known to produce localized DNA double-strand breaks. If these breaks are potentially lethal lesions, they must be repaired, since the mean lethal dose (D₀) was 28 dpc. The observations that a single dpc has a high probability of producing a mutation (mutagenic efficiency 1.0–3.0) would suggest, however, that this repair is extremely error-prone. If the breaks need not be repaired to permit survival, then lethal lesions are a subset of or are completely different from mutagenic lesions.     

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.     

Induction of specific-locus and dominant lethal mutations in male mice by 1-methyl-1-nitrosourea (MNU)

1-Methyl-1-nitrosourea (MNU) induced specific-locus mutations in mice in all spermatogenic stages except spermatozoa. After intraperitoneal injection of 70 mg/kg body weight of MNU a high yield of specific-locus mutations was observed in spermatids (21.8 × 10⁻⁵ mutations per locus per gamete). The highest mutational yield was induced in differentiating spermatogonia. In 1954 offspring we observed 5 specific-locus mutants (44.8 × 10⁻ mutations per locus per gamete). In addition, 2 mosaics were recovered, which gave a combined mutation rate of 62.7 × 10⁻⁵. In A_s spermatogonia the mutation rate was 3.9 × 10⁻⁵. The same dose of 70 mg/kg of MNU induced dominant lethal mutations 5–48 days post treatment, mainly due to post-implantation loss in spermatids and spermatocytes. It is interesting to compare the induction pattern of mutations by MNU with methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and ethylnitrosourea (ENU). Based on the different spermatogenic response of the induction of specific-locus mutations we can characterize the 4 mutagens in the following way: EMS = MMS ≠ MNU ≠ ENU.     

UV-induced mutagenesis at the hypoxanthine—guanine phosphoribosyl transferase locus in two L5178Y mouse lymphoma cell strains with different UV sensitivities

Two strains of L5178Y mouse lymphoma cells, L5178Y-R (LY-R) and L5178Y-S (LY-S), differ markedly in their sensitivity to 254 nm UV radiation (D₀ = 0.7 and 5.5 J/m²; n = 6.0 and 2.0 for LY-R and LY-S cells, respctively). In this study, the frequency o hypoxanthine-guanine-phosporibosyl-transferase-deficient mutants was determined, using 6-thioguanine (TG) as a selective agent, in populations of LY-R and LY-S cells exposed to various fluences of UV radiation. The spontaneous mutation frequency for LY-R cells was (3.7 ± 0.6) × 10^?5 TG^r mutants per viable cell, and the UV induction rate was (2.2 ± 0.8) × 10^?4 TG^r mutants per viable cell, per J/m². Both spontaneous and induced mutantion frequencies were much lower for LY-S cells. The sopntaneous mutation frequency for these cells were too low to make its measurement practicable ( < 0.0013 × 10^?5 TG^r mutants per viable cell). Mutation induction rate was (4.2 ± 2.2) × 10^?7 TG^r mutants per viable cell, per J/m². These differences in mutability do not appear to be due to gene duplication in LY-S cells, or to selective growth disadvantage of LY-S-derived TG-resistant mutants. Possible mechanisms underlying the differences in mutability of LY-R and LY-S cells are considered.     

Influence of anoxia and respiratory deficiency on the genotoxicity of some direct-acting alkylating agents in yeast

We have studied the influence of anoxia and respiratory deficiency (RD) in yeast on the cytotoxic and recombinogenic effects of 5 direct-acting alkylating agents, namely N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), methylnitrosourea (MNU), ethylnitrosourea (ENU), methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS). We found that the effects of both conditions parallel each other for MMS, MNNG, MNU and ENU. Both anoxia and RD did not modify the effects of MMS to any significant extent. On the other hand, anoxic and respiratory-deficient cells were found to be more resistant than euoxic and respiratory-proficient cells respectively for MNNG, MNU and ENU. In the case of EMS, which is similar to MMS in its chemical reaction with DNA, the respiratory-deficient cells were found to be more sensitive than the respiratory-proficient ones. These studies indicate that the response of anoxic and respiratory deficient cells cannot be predicted solely on the basis of the chemical reactivity pattern of the alkylating agents. The physiological state which exists under these conditions may exert considerable influence on the cellular response.     

The relationship between reaction kinetics and mutagenic action of monofunctional alkylating agents in higher eukaryotic systems. IV. The effects of the excision-defective mei-9L1 and mus(2)201D1 mutants on alkylation-induced genetic damage in Drosophila

Repair-defective mutants of Drosophila melanogaster which identify two major DNA excision repair loci have been examined for their effects on alkylation-induced mutagenesis using the sex-linked recessive lethal assay as a measure of genotoxic endpoint. The alkylating agents (AAs) chosen for comparative analysis were selected on the basis of their reaction kinetics with DNA and included MMS, EMS, MNU, DMN, ENU, DEN and ENNG. Repair-proficient males were treated with the AAs and mated with either excision-defective mei-9L1 or mus(2)201D1 females or appropriate excision-proficient control females. The results of the present work suggest that a qualitative and quantitative relationship exists between the nature and the extent of chemical modification of DNA and the induction of of genetic alterations. The presence of either excision-defective mutant can enhance the frequency of mutation (hypermutability) and this hypermutability can be correlated with the Swain-Scott constant S of specific AAs such that as the SN1 character of the DNA alkylation reaction increases, the difference in response between repair-deficient and repair-proficient females decreases. The order of hypermutability of AAs with mei-9L1 relative to mei-9+ is MMS greater than MNU greater than DMN = EMS greater than iPMS = ENU = DEN = ENNG. When the percentage of lethal mutations induced in mei-9L1 females are plotted against those determined for control females, straight lines of different slopes are obtained. These mei-9L1/mei-9+ indices are: MMS = 7.6, MNU = 5.4, DMN = 2.4, EMS = 2.4 and iPMS = ENU = DEN = ENNG = 1. An identical order of hypermutability with similar indices is obtained for the mus(2)201 mutants: MMS(7.3) greater than MNU (5.4) greater than EMS(2.0) greater than ENU(1.1). Thus, absence of excision repair function has a significant effect on mutation production by AAs efficient in alkylating N-atoms in DNA but no measurable influence on mutation production by AAs most efficient in alkylating O-atoms in DNA. The possible nature of these DNA adducts has been discussed in relation to repair of alkylated DNA. In another series of experiments, the effect on alkylation mutagenesis of mei-9L1 was studied in males, by comparing mutation induction in mei-9L1 males vs. activity in Berlin K (control). Although these experiments suggested the existence of DNA repair in postmeiotic cells during spermatogenesis, no quantitative comparisons could be made.(ABSTRACT TRUNCATED AT 400 WORDS)     

Critical periods of the mitotic cycle: influence of aminopterin and thymidine on production of chromosomal aberrations by radiation in Crepis capillaris.

The influence of aminopterin (AP), tritiated thymidine ([3H] TdR) and "cold" thymidine (TdR) on production of chromosomal aberrations in meristematic cells of Crepis capillaris irradiated in different stages of the mitotic cycle with 300 rad of 63Co gamma-rays was studied. All the chemical treatments increased most of all the frequency of aberrations induced during two "critical periods" localized before the stage of DNA synthesis (fixation 9 h after irradiation) and before that of mitosis (4 h). Treatments with TdR and [3H]TdR increased most of all the frequency of chromatid aberrations when irradiation was performed in G1, and the frequency of gaps when irradiated in G2. Treatment with AP increased the yield of different types of aberration more uniformly. The modifying effect of the chemicals tested appeared to be independent of replicative synthesis. The "critical periods" are suggested to be the stages when regular "proof reading" and correction of spontaneous errors takes place [9,13]. In addition to this regular mechanism, radiation induces an "emergency" mechanism of repair. AP inhibits the mechanism of regular repair; in addition TdR and [3H] TdR suppress the lateral spread of primary injuries across the chromosome.     

Enhancement by caffeine of N-methyl-N-nitrosourea-induced mutations and chromosome aberrations in Chinese hamster cells

N-Methyl-N-nitrosourea (MNU) increased the induction of mutations to 8-azaguanine resistance in Chinese hamster cells in a dose-dependent manner. Mutations were only observed with toxic concentrations of MNU. Since a plot of the fraction of cells surviving alkylation against the extent of methylation of DNA exhibited a shoulder it followed that there was a threshold level of DNA reaction which did not lead to mutations possibly due to efficient repair of DNA damage. Post-alkylation incubation in medium containing caffeine decreased cell survival while at the same time it increased the induced mutation frequency. Mutation frequency was increased whether caffeine was present for 48 h or for a further 12 days in the presence of the selective agent 8-azaguanine. MNU caused chromatid aberrations in Chinese hamster cells and these reached a value of 15% of the treated cells by 48 h after methylation. Post-alkylation incubation in caffeine increased the percentage of cells showing chromosomal damage to a maximum of 86% of treated cells by 40 h after alkylation. A large proportion of cells exhibited completely fragmented or shattered chromosomes. The proportion of cells showing the presence of micronuclei also dramatically increased following incubation of methylated cells in caffeine. These results are discussed in terms of the possibility that damage to DNA is responsible for the lethal, mutagenic and cytological effects of MNU in Chinese hamster cells, and that there is a caffeine sensitive step(s) in the repair of the DNA damage which is responsible for these effects.     

Effect of cycloheximide on protein and DNA synthesis and on the yields of chromosomal aberrations induced by DEB and ENU in vicia faba

Incubation of root tips in cycloheximide (CHM) at concentrations of 0.3–50 μg/ml inhibits the incorporation of [¹⁴C]leucine by 40–100% within 2 h. A depression in the incorporation of [³C]thymidine was observed after a 2-h incubation in CHM solution at 1 μg/ml.In root tips exposed for 2 h to CHM at 1 μ/ml the mitotic activity of cells was severely depressed within 15 h of recovery. Metaphases appearing after 20 h carried infrequent aberrations of the chromatid type. CHM at this concentration had no effect on the yield of aberrations induced by the alkylating agents diepoxybutane (DEB) and N-ethyl-N-nitrosourea (ENU) when applied as post-treatment.     

The effect of thymidine on the induction of micronuclei by alkylating agents in V79 Chinese hamster cells

Incubation in thymidine-containing medium resulted in increased lethality and micronucleus frequency in V79 cells treated with ethyl nitrosourea (ENU), methyl nitrosourea (MNU) and ethyl methanesulphonate (EMS) but not with methyl methanesulfonate (MMS). Thymidine had no effect in ENU treated HeLa cells. In V79 cells, the presence of thymidine during post-treatment DNA replication was necessary for the effect. It is suggested that the increase in chromosome damage was the result of an increased O⁶-alkylguanine-thymine mispairing in cells which are defective in the repair of O⁶-alkylguanine. Treatment of V79 cells with O⁶-ethylguanine resulted in increased production of both micronuclei and polyploid cells. These effects might be explained by spindle dysfunction caused by the alkylated guanine.     

Facilitation by pyrimidine deoxyribonucleosides and hypoxanthine of mutagenic and cytotoxic effects of monofunctional alkylating agents in Chinese hamster cells.

Hypoxanthine (Hx), thymidine (TdR) and deoxycytidine (CdR), at concentrations of 10(-5) M increased the yield of 8-azaguanine-resistant (AzGr) mutants induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in cultured Chinese hamster V79 cells. The cytotoxicity of MNNG was increased 2-fold in the presence of Hx, and more than 10-fold in the presence of TdR. This cytotoxic effect of TdR was abolished by equal concentrations of CdR, which by itself did not increase the cytotoxicity of MNNG. However, the yield of MNNG-induced AzGr colonies was increased 2--10-fold in the presence of both CdR and TdR. The AzGr colonies displayed phenotypes characteristic of hypoxanthine: guaninephosphoribosyltransferase-deficient (HGPRT-) mutants, or indicative of mutant HGPRT with altered substrate affinities. The nucleosides did not affect the growth or expression time of the HGPRT- mutants; the same extent of alkali-labile DNA damage occurred in cells treated with alkylating agents in the presence and absence of TdR and CdR; and the increase in mutation frequency in the presence of these nucleosides occurred not only with MNNG, but also with ethylating agents. Nucleosides treated with MNNG were not mutagenic, and treatment of the cells with TdR and CdR only prior to treatment with MNNG or only during selection with AzG did not increase the induced mutation frequency. Therefore, the interpretation is proposed that CdR, TdR and Hx produce nucleotide-pool imbalances that increase lethal and mutagenic errors of replication of alkylated DNA.     

Use of a T-lymphocyte clonal assay for determining HPRT mutant frequencies in individual rats

Conditions for detection and isolation of HPRT⁻ mutants in cloned ratT-lymphocytes from individual adult Lewis rats were determined. Similar to cloning of human T-cells, best results were obtained with lectin (PHA)-primed T-lymphocytes of rats. High cloning efficiencies, occasionally exceeding 50%, could be obtained when the target cells employed were isolated from cervical lymph nodes. Feeder cells used were splenocytes, irradiated with 40 Gy of X-rays after priming with Con A. Human interleukin-2, present in LAK supernatant, proved to be capable of inducing proliferative activity of rat T-lymphocytes and could replace conditioned medium from primed rat splenocytes.Under the conditions described in this paper, the frequency of mutants in the HPRT gene of T-lymphocytes in Lewis rats was about 80% lower than that found in human T-lymphocytes from adults. The inverse relationship between mutant frequency and cloning efficiency, clearly demonstrated for human data, could not be established for rats. Treatment of rats with N-ethyl-N-nitrosourea, a potent alkylating agent, resulted in a time- and dose-dependent induction of HPRT⁻ mutants, demonstrating the usefulness of this system to study in vivo mutagenesis.     

Alkylation of transfer RNA by N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea

The alkylation of a number of purified tRNA preparations by reaction with the carcinogens, N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea was studied in order to investigate the role of nucleic acid structure on the distribution of alkylation products within the nucleotide sequence. The rate of alkylation was greatly increased by increasing the pH over the range 6 to 8 and the degree of alkylation (expressed as moles alkyl groups/mole tRNA) was directly proportional to the concentration of the nitrosamide added and independent of the amount of tRNA present. There was no significant difference in the degree of alkylation of any of the tRNA preparations tested. Reaction with N-ethyl-N-nitrosourea resulted in a degree of alkylation some 13 times less than that produced by reaction with a similar concentration of N-methyl-N-nitrosourea. The major product of the reaction was 7-alkylguanine amounting to about 80% of the total, but 3-methylcytosine, 6-O-methylguanine and 1-methyl-, 3-methyl-, and 7-methyladenine were also identified as products of the reaction of tRNA^fMet with N-methyl-N-nitrosourea.The possibility that preferential alkylation of certain residues within the polynucleotide sequence was produced by reaction with the nitrosamides was examined by degradation of the alkylated tRNA with pancreatic ribonuclease and separation of the oligonucleotide fragments by chromatography on DEAE cellulose. When tRNA^fMet which had been alkylated by reaction with N-methyl-N-nitrosourea or N-ethyl-N-nitrosourea was analysed in this way, the distribution of 7-alkylguanine was, within the limits of experimental error, in agreement with that expected for a random reaction of the alkylating agent with all of the guanosine residues throughout the molecule. A similar result was seen when tRNA^Phe was examined. These results were obtained by alkylation under conditions where the native configuration of the tRNA was maintained and show that the tertiary structure of the nucleic acid does not impart any specificity to the reaction with the nitrosamide producing 7-alkylguanine but the possibility that such specificity does exist for the minor products of alkylation cannot be excluded.     

Bladder cancer and N-methyl-N-nitrosourea. II. Sub-cellular changes associated with a single noncarcinogenic dose of MNU

The ultrastructural changes in the rat urothelium induced by a single, cytotoxic but not carcinogenic dose of N-methyl-N-nitrosourea(MNU) are described. They are compared with some aspects of the cyclophosphamide-induced response.Both MNU and cyclophosphamide produce cell necrosis, desquamation of the epithelium and haemorrhage from sub-epithelial capillaries, followed by epithelial hyperplasia. The hyperplastic epithelium appears to be de-differentiated, but the effect is reversible, and a normal-looking epithelium is re-established in 10 to 15 weeks after the MNU treatment.The significance of breaks in the basal lamina in this situation is discussed and the phagocytic potential of the epithelial cells is illustrated.