Specificity of mutagenic effect of N-nitroso-N-methylurea and relation between its mutagenic activity and carbamoylating properties

Significance of carbamoylation for mutagenic effects of N-nitroso-N-methyl-urea (NMU) on the CHO-AT3-2 cell line of Chinese hamster was studied. True point mutations occurred, due to alkylation. Carbamoylation combined with alkylation, or carbamoylation after alkylation induced the increase in other types of gene mutations as well as micro- and macroaberrations. These effects may be explained by the synergistic effect of alkylation and carbamoylation. Possible mechanisms and levels of interaction between alkylation and carbamoylation are discussed.     

Role of the carbamoylation reaction in the biological activity of methyl nitrosourea

Study of the mutagenic action of methyl nitrosourea (MNU) on the CHO-AT3-2 Chinese hamster cell at 2 regimes of cell treatment (a short-term regime and prolonged 1-h treatment) revealed that increase in the duration of treatment enhanced both cell lethality and clastogenic and mutagenic effects at the TK locus and did not influence the mutation frequency at the OUAr locus. On the basis of kinetic considerations it can be concluded that the base-pair substitution-type mutants (e.g., OUAr) appear as a result of DNA alkylation and the mutants at loci with a wide spectrum of registered mutants (the TK locus) are related to a greater extent to the carbamoylating activity of MNU. This conclusion is confirmed by measurements of the effects of sequential treatment with MNU (7 min) and KNCO (1 h). A synergistic increase in lethality, clastogenicity and mutagenicity at the TK locus was found in experiments with the combined treatment of cells with ethyl methanesulfonate (EMS) and KNCO. Besides, pretreatment of cells with potassium cyanate and subsequent exposure to MNU, EMS and benzopyrene (BP) produced synergistic effects in all the tests: lethality, clastogenicity and mutation frequency at the OUAr and TK loci. Posttreatment of cells with KNCO also led to a synergistic increase in the effects of MNU, EMS and BP treatment in several tests, but not in the OUAr locus. The possible mechanism and levels of interactions between alkylation and carbamoylation and the possibility that potassium cyanate causes supramolecular lesions are discussed.     

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.     

Reduction in the frequency of N-methyl-N-nitrosourea-induced somatic mutations in Tradescantia by pretreatment with low doses of alkylating agents

Treatment of Tradescantia cuttings with sub-mutagenic doses of N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea and methyl methanesulphonate before challenging doses of MNU reduced the frequency of somatic mutations in stamen hairs as compared with the effect of challenging dose alone. The highest response was about a 50% reduction in the mutagenic effect of the challenge dose.     

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.     

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.     

Comparison of the genetic effects of equimolar doses of ENU and MNU: while the chemicals differ dramatically in their mutagenicity in stem-cell spermatogonia, both elicit very high mutation rates in differentiating spermatogonia

Mutagenic, reproductive, and toxicity effects of two closely related chemicals, ethylnitrosourea (ENU) and methylnitrosourea (MNU), were compared at equimolar and near-equimolar doses in the mouse specific-locus test in a screen of all stages of spermatogenesis and spermiogenesis. In stem-cell spermatogonia (SG), ENU is more than an order of magnitude more mutagenic than MNU. During post-SG stages, both chemicals exhibit high peaks in mutation yield when differentiating spermatogonia (DG) and preleptotene spermatocytes are exposed. The mutation frequency induced by 75mgMNU/kg during this peak interval is, to date, the highest induced by any single-exposure mutagenic treatment - chemical or radiation - that allows survival of the exposed animal and its germ cells, producing an estimated 10 new mutations per genome. There is thus a vast difference between stem cell and differentiating spermatogonia in their sensitivity to MNU, but little difference between these stages in their sensitivity to ENU. During stages following meiotic metaphase, the highest mutation yield is obtained from exposed spermatids, but for both chemicals, that yield is less than one-quarter that obtained from the peak interval. Large-lesion (LL) mutations were induced only in spermatids. Although only a few of the remaining mutations were analyzed molecularly, there is considerable evidence from recent molecular characterizations of the marker genes and their flanking chromosomal regions that most, if not all, mutations induced during the peak-sensitive period did not involve lesions outside the marked loci. Both ENU and MNU treatments of post-SG stages yielded significant numbers of mutants that were recovered as mosaics, with the proportion being higher for ENU than for MNU. Comparing the chemicals for the endpoints studied and additional ones (e.g., chromosome aberrations, toxicity to germ cells and to animals, teratogenicity) revealed that while MNU is generally more effective, the opposite is true when the target cells are SG.     

Effect of alkylation and carbamoylation on the characteristics of genetic damage to mammalian somatic cells cultured in vitro

Genetic effects of alkylation alone and combined with carbamoylation were studied following treatment of CHO-AT3-2 Chinese hamster cell line with N-nitroso-N-methylurea for 7 and 60 min. Gene mutations at HGPRT and Na+/K+-ATPase loci, micronuclei, cells with fragmented nuclei and lethality caused by NMU were recorded. Prolonged exposure to the mutagen made these effects more pronounced, particularly the fragmented nuclei and cell death. The combined action of the two mechanisms, therefore, enhanced the mutagenic effects of alkylation and expanded the range of DNA lesions towards greater incidence of gross damage to chromosomes and chromatids.     

Spontaneous and induced chromosomal aberrations and gene mutations in human lymphoblasts: mitomycin C, methylnitrosourea, and ethylnitrosourea

The concentration-dependent mutagenic, clastogenic, and cytocidal activities of mitomycin C (MC), methylnitrosourea (MNU), and ethylnitrosourea (ENU) were measured in the human lymphoblast cell line TK6. For treatments resulting in fewer than 2 lethal hits, MNU, ENU, and MC gave rise to apparently linear dose-response curves for gene mutations (hgprt and tk genes) as well as for chromosomal aberrations. The numbers of induced mutants at the tk and hgprt loci were similar between the two loci for each compound. However, the ratio of mutagenic activity relative to the clastogenic activity (aberrations/cell) was lowest for mitomycin C, intermediate for methylnitrosourea, and highest for ethylnitrosourea. These results confirm in human cells the general observation that the processes of mutagenesis and clastogenesis are nonidentical: compounds vary independently in their mutagenic and clastogenic potentials.     

O-alkylation in DNA does not correlate with the formation of chromosome breakage events in D. melanogaster

Postmeiotic cell stages of repair-proficient ring-X (RX) males were treated with methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), diethylnitrosamine (DEN) or ethylnitrosourea (ENU) and then mated to either repair-defective (mei-9L1) or to repair-competent females (mei-9+). Absence of the mei-9+ function resulted in a hypermutability effect to all alkylating agents (AAs) when they were assayed for their ability to induce chromosomal aberrations (chromosome loss; CL), irrespective of marked differences in distribution of DNA adducts brought about by these AAs. This picture is different from that described previously for the induction of point mutations (Vogel et al., 1985a). There, evidence was presented indicating that reduction in DNA excision repair does not affect point mutation induction (recessive lethals) by those AAs most efficient in ring-oxygen alkylation such as ENU, DEN, N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), and isopropyl methanesulfonate (iPMS): the order of hypermutability of AAs with mei-9L relative to mei-9+ was 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 were plotted against those determined for mei-9+ females, straight lines of following slopes were obtained: MMS = 7.6, MNU = 5.4, DMN = 2.4, EMS = 2.4, and iPMS = ENU = DEN = ENNG = 1. Those findings, together with the recent observation that AAs do not split into two groups when assayed for their ability to cause CL, point to the involvement of different DNA alkylation products in ENU- and DEN-induced chromosome loss vs. that of point mutations. It is concluded that with ENU and DEN chromosomal loss results from N-alkylation products whereas point mutations (SLRL) are the consequence of interactions with oxygen-sites in DNA. Thus, as a consequence of a very dominating role of O-ethylguanine (and possibly O4-alkylation of thymine), N-alkylation in DNA does not contribute measurably to mutation induction in the case of ENU-type mutagens while O-alkylation, very clearly, does not show a positive correlation with the formation of chromosome breakage events in Drosophila. Conversely, it appeared that with MMS-type mutagens (MMS; dimethyl sulfate, DMS; trimethyl phosphate, TMP), alkylation products such as 7-methylguanine and 3-methyladenine, if unrepaired or misrepaired, are potentially mutagenic lesions causing both mutations and chromosomal aberrations.     

Deoxyribonucleotide pools as targets for mutagenesis by N-methyl-N-nitrosourea

This paper describes a biological test of the hypothesis that one or more components of the intracellular nucleotide pool represent a significant target for the mutagenic effects of alkylating agents. In other words, we ask whether mutagenesis can occur either through alkylation of susceptible nucleotide residues in DNA, or through alkylation of a free nucleotide, followed by its incorporation into DNA. Our approach is based upon the premise that if a nucleotide pool is a mutagenic target, then transient expansion of that pool should increase the target size and enhance mutagenesis following subsequent treatment with an alkylating target.

Working either with V79 hamster lung fibroblasts or Chinese hamster embryo fibroblasts (CHEF/18), we treated cells for 30 min, under conditions that expanded one or more pools of deoxyribonucleoside triphosphates. This was followed immediately by a 30-min treatment with 0.5 mM N-methyl-N-nitrosourea. After 8 days of additional culture for recovery of cells and expression of mutations, we plated in selective media to determine the abundance of 6-thioguanine-resistant mutants in each culture.

We found that conditions which expand pools of either dATP or dTTP and dGTP stimulate mutagenesis by MNU, with the degree of stimulation varying in different experiments from 2-to 6-fold. Although alternate interpretations can be entertained, the data are consistent with the hypothesis that nucleotide pools represent alkylation targets. A biochemical test of the hypothesus us warranted.

During our studies we made several other noteworthy observations: (1) treatment of V79 cells with mutagen alone does not significantly affect dNTP pools; (2) deoxynucleotide pool perturbations are quite short-lived following transfer of cells to normal medium; (3) deoxyuridine is significantly more effective than thymidine in expanding dTTP pools; (4) deoxyuridine by itself is significantly mutagenic, particularly to CHEF/18 cells.     

Aphidicolin allows a rapid and simple evaluation of DNA-repair synthesis in damaged human cells

The decrease in microbial mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (MNU) was compared in an animal mediation with rats and in direct incubation with human as well as rat blood and blood components. The mutagenic activity was assayed by reverse mutation from streptomycin (SM) dependence to non-dependence in Escherichia coli, strain Sd-B (TC). The mutagenic response curves of both MNNG and MNU were approximately linear and parallel at non-cytotoxic concentrations. However, the mutagenic capabilities of MNNG were estimated to be 10-fold more potent than those of MNU. The mutagenic activity in blood and liver preparations from rats killed immediately after intravenous injection of MNNG, 50 mg/kg, was negative. Results with MNU, 100 mg/kg, were positive in both cases.For the detection of mutagenicity, blood was diluted 50 times for the final testing mixture (1 ml) to avoid bactericidal effects of the blood itself. When a larger amount of liver preparation was used in the tests, and diluted 8 times, mutagenic activity was still detected 15 min after injection of MNU, 80 mg/kg. Comparisons of the diminished rate of mutagenicity between MNNG and MNU during certain periods of incubation with blood indicated that MNNG was inactivated much more rapidly than MNU with both human and rat blood. Plasma showed a moderate inactivating effect on both MNNG and MNU. Red blood cells inactivated MNNG at a remarkably rapid rate similar to that of whole blood, but was less effective on MNU. In further experiments with red- cell components, the cell contents inactivated both MNNG and MNU at rates similar to those with red cells, but cell membrane had absolutely no effect in decreasing the mutagenicity in either MNNG or MNU.     

The elimination of mutagenic lesions induced by alkylating agents and UV in V79 Chinese hamster cells

The mutagenicity of MNU, EMS, BMS and UV light was compared by analyzing the dose-response curve just before and after the replicative process of the HGPRT gene in synchronized V79 Chinese hamster cells. This system makes it possible to compare a 10-h period for repair of different mutagenic lesions with no time for repair. Additional time for repair in synchronized V79 cells resulted in a reduced response for MNU and UV, but not for EMS and BMS. This result suggests that an error-free repair process operates on mutagenic lesions in methylated DNA and on thymine dimers, but not on ethylated and butylated DNA. Based on these results, it is concluded that the repair capacity of V79 cells to remove mutagenic lesions is characterized as low for UV, moderate for MNU and not detectable for the mutagenic lesions induced by EMS and BMS.     

Comparative mutagenicity of chemicals selected for test in the International Program on Chemical Safety''s collaborative study on plant systems for the detection of environmental mutagens

A review has been made of the four compounds (maleic hydrazide, methyl nitrosourea, sodium azide, azidoglycerol) tested in the International Program on Chemical Safety's collaborative study systems. Maleic hydrazide (MH) is a weak cytotoxic/mutagenic chemical in mammalian tissues and is classified as a class 4 chemical. In contrast, with few exceptions such as Arabidopsis, MH is a potent mutagen/clastogen in plant systems. The difference in its response between plant and animal tissue is likely due to differences in the way MH is metabolized. MH appears to be noncarcinogenic and has been given a negative NCI/NTP carcinogen rating.

Methyl nitrosourea (MNU) is a toxic, mutagenic, radiomimetic, carcinogenic, and teratogenic chemical. It has been shown to be a mutagen in bacteria, fungi, Drosophila, higher plants, and animal cells both in vitro and in vivo. MNU is a clastogen in both animal and human cell cultures, plant root tips and cell cultures inducing both chromosomes and chromatid aberrations as well as sister-chromatid exchanges. Carcinogenicity has been confirmed in numerous studies and involves the nervous system, intestine, kidney, stomach, bladder and uterus, in the rat, mouse, and hamster. MNU produces stage-specific teratogenic effects and also interferes with embryonic development. The experimental evidence that strongly indicates the mutagenic effects of MNU underlines the possible hazard of this compound to human beings. The experimental evidence for the stringent handling of this compound is clear.

Sodium azide (NaN₃) is cytotoxic in several animal and plant systems and functions by inhibiting protein synthesis and replicative DNA synthesis at low dosages. It is mutagenic in bacteria, higher plants and human cells and has been used as a positive control in some systems. In general, tests for clastogenicity have been negative or weakly positive. No evidence of carcinogenicity has been reported in a 2-year study seeking carcinogenic activity in male and female rats. Its advantages in comparison to other efficient mutagens are claimed to be a high production of gene mutations accompanied by a low frequency of chromosomal rearrangements and safer handling because of its nonclastogenic and noncarcinogenic action on humans.     

Selective effect of p-aminobenzoic acid on mutagenesis: phenotypic analysis of the Arg+-revertants induced by N-nitroso-N-methylurea in the Escherichia coli K-12 AB1157 strain

According to the phenotypic analysis of Arg+ revertants in Escherichia coli K-12 AB1157, the specific mutational changes in bacterial cells under the action of MNU were registered. True and suppressor mutations of four phenotypic groups were noted. The quantity of mutants induced depended upon the DNA and protein syntheses in bacterial cells. The sublethal concentration of para-aminobenzoic acid markedly (10-50 fold) reduced the rate of mutagenic induction and changed the quantity relations of mutants within phenotypic groups.     

Enhanced mutagenic response of MNU by post-treatment with methylmercury,caffeine or thymidine in V79 Chinese hamster cells

Post-treatment with a non-toxic dose of thymidine, caffeine or methylmercury-hydroxide enhanced the mutagenic response from MNU at high doses, whereas no enhancement was observed with low doses of MNU. No mutagenic effects were found with thymidine, caffeine or methylmercury alone at the doses used. Quantitatively, the modifying effects observed were similar for the 3 agents which indicates the possibility of a similar way of action.     

Cross-resistance studies with V79 Chinese hamster cells adapted to the mutagenic or clastogenic effect of N-methyl-N′-nitro-N-nitrosoguanidine

When V79 cells are exposed to a single low dose of MNNG or MNU they acquire resistance to the mutagenic or to the clastogenic effect of the agents. Here the effect of MNNG pretreatment on mutagenesis (6-thioguanine resistance) and aberration formation in cells challenged with various mutagens/clastogens is reported. MNNG-adapted cells were resistant to the mutagenic effects of MNU and, to a lower extent, of EMS. No mutagenic adaptation was observed when MNNG-pretreated cells were challenged with MMS, ENU, MMC or UV.

Cells pretreated with a dose of MNNG which makes them resistant to the clastogenic effect of this compound were also resistant to the clastogenic activity of other methylating agents (MNU, MMS), but not so with respect to ethylating agents (EMS, ENU). Cycloheximide abolished the aberration-reducing effect of pretreatment. However, when given before the challenge dose of MNNG, MNU or MMS, it drastically enhanced the aberration frequency in both pretreated and non-pretreated cells. No significant enhancement of aberration frequency by cycloheximide was found for ethylating agents.

The results indicate that clastogenic adaptation is due to inducible cellular functions. It is concluded that mutagenic and clastogenic adaptation are probably caused by different adaptive repair pathways.     

Alkylation damage causes MMR-dependent chromosomal instability in vertebrate embryos

S(N)1-type alkylating agents, like N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU), are potent mutagens. Exposure to alkylating agents gives rise to O(6)-alkylguanine, a modified base that is recognized by DNA mismatch repair (MMR) proteins but is not repairable, resulting in replication fork stalling and cell death. We used a somatic mutation detection assay to study the in vivo effects of alkylation damage on lethality and mutation frequency in developing zebrafish embryos. Consistent with the damage-sensing role of the MMR system, mutant embryos lacking the MMR enzyme MSH6 displayed lower lethality than wild-type embryos after exposure to ENU and MNU. In line with this, alkylation-induced somatic mutation frequencies were found to be higher in wild-type embryos than in the msh6 loss-of-function mutants. These mutations were found to be chromosomal aberrations that may be caused by chromosomal breaks that arise from stalled replication forks. As these chromosomal breaks arise at replication, they are not expected to be repaired by non-homologous end joining. Indeed, Ku70 loss-of-function mutants were found to be equally sensitive to ENU as wild-type embryos. Taken together, our results suggest that in vivo alkylation damage results in chromosomal instability and cell death due to aberrantly processed MMR-induced stalled replication forks.