DNA repair and chromosomal stability in the alkylating agent-hypersensitive Chinese hamster cell line 27-1

27-1 is a mutant of Chinese hamster ovary cells (CHO cells) that is hypersenstivie to the toxic effects of ultraviolet light, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and other monofunctional alkylating agents. We show here that the enhanced MNNG sensitivity of these cells is not due to alterations in the amount of DNA methylation products introduced nor by a defect in the first step of removal of the main alkylation products 7-methylguanine and 3-methyladenine. However, these mutant cells perform more DNA repair synthesis after treatment with MNNG than normal CHO-9 cells. This observation might indicate a possible defect of a ligase involved in sealing DNA repair patches.27-1 cells did not show elevated frequencies of sister-chromatid exchange and chromosomal aberration induced by MNNG. The data show that MNNG-induced cell killing is not necessarily related to increased chromosomal instability.     

Mechanism of potent mutagenic action of N-methyl-N'-nitro-N-nitrosoguanidine on intracellular phage lambda.

N-Methyl-N′-nitro-N-nitrosoguanidine efficiently induces mutations from “clear” to “virulent” in phage λ only during the intracellular growth phase. Lambda DNA extracted from infected bacteria after treatment with MNNG³ produced a mutant yield about 100-fold higher than the spontaneous level upon transfection of MNNG-treated spheroplast cells, whereas the yield diminished an order of magnitude when assayed on untreated spheroplasts. As measured by ¹⁴C incorporation after treatment with [methyl-¹⁴C]MNNG, λ DNA packed in head protein was methylated to about 3% by an MNNG dose of 0.6 mg/ml but was barely mutagenised; whereas intracellular λ DNA was methylated to no more than 0.6% by an MNNG dose of 0.09 mg/ml and was highly mutagenised. Lambda phages treated in vitro with ethyl methanesulfonate produced a rather low mutant yield on untreated cells but the yield increased about tenfold on MNNG-treated cells. Mutability of untreated λ on cells having received an F′ factor was enhanced efficiently by ultraviolet light, but not so by MNNG, previously applied to the F′. Surprisingly similar MNNG dose-effect curves exist for enhancing spontaneous, mispairing (MNNG or EMS induced) and misrepair (ultraviolet light induced) mutagenesis of λ. From these and other data we conclude that MNNG hypermutagenesis results from a synergistic increase in mispairing probability of appropriately methylated bases (by action of MNNG in vivo) in the target gene within an MNNG-induced intracellular environment that has an enhanced mutagenic capacity.     

Response of human keratinocytes to extremely low concentrations of N-methyl-N′-nitro-N-nitrosoguanidine

Since alkylating agents are widely present in the environment and constitute a continuous challenge to genome integrity, cells and organisms have developed defense mechanisms to remove such lesions. We monitored the response of human keratinocytes to a very low concentration of a methylating agent, namely 2.5 nM N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). The effect of a 60-min exposure of quiescent cells to 2.5 nM MNNG was studied in terms of DNA integrity, poly(ADP-ribose) metabolism, clonogenic survival and DNA synthesis. We observed two waves of DNA strand break formation and resealing. Interestingly, the amount of DNA strand breaks in exposed cells was lower than in unexposed control cells. This phenomenon was also observed when cells were exposed to MNNG in the presence of a protein synthesis inhibitor, or when they were maintained on ice during the treatment. A dose of 2.5 nM MNNG stimulated poly(ADP-ribose) turnover, reduced the intracellular NAD⁺ content, stimulated DNA synthesis and caused a remarkable increase in clonogenic survival. Thus, the cellular responses to extremely low concentrations of MNNG differ sharply from those observed at higher doses of this carcinogen. We conclude that the very low dose response cannot be extrapolated from usual dose-response analyses.     

5-Methyltryptophan resistance mutations in Escherichia coli K-12. Mutagenic activity of monofunctional alkylating agents including organophosphorus insecticides

The induction of 5-methyltryptophan (5-MT) resistance mutations was assayed as a test system for mutagenic chemicals in Escherichia coli. It is assumed that different premutational alterations in several genes of the Escherichia coli chromosome will lead to 5-MT-resistant mutants. The chemicals used were three monofunctional alkylating agents as reference compounds, namely β-propiolactone (β-PL), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), and methyl methanesulfonate (MMS), which are all mutagenic in the 5-MT system; of the eight organophosphorus insecticides tested, four have definite mutagenic activity (Dichlorvos, Oxydemetonmethyl, Dimethoate, and Bidrin), one is probably mutagenic (Methylparathion) and the remaining three (Parathion, Malathion and Diazinon) do not induce 5-MT resistance mutations in the conditions used here (< 30% survival). The relative mutagenic activity after a treatment time of 60 min is (in decreasing order) MNNG > MMS > Dichlorvos > Oxydemetonmethyl, Dimethoate and Bidrin. The concentration-dependent mutagenic activity of all mutagenic compounds is nearly linear when plotted on a log-log scale (with slopes varying from 1.0 to 1.5) and could be taken as an indication that one premutational reaction will be sufficient for the induction of one 5-MT-resistant mutant.     

The effect of caffeine upon cell survival and post-replication repair of DNA after treatment of BHK 21 cells with either UV irradiation or N-methyl-N-nitrosoguanidine

It has been found that in BHK 21 cells caffeine potentiates cell killing by both UV irradiation and N-methyl-N-nitrosoguanidine (MNNG). The potentiating effect is greater with UV than with MNNG. While non-toxic concentrations of caffeine inhibit the joining of newly-replicated DNA fragments into large molecular weight DNA (post-replication repair) after UV irradiation, they have no such effect after MNNG treatment. Furthermore, the joining of DNA fragments continues in cells treated with 3 μg/ml of MNNG, a dose which leads to less than 5% cell survival. While inhibition of the synthesis of large molecular weight DNA can explain the synergistic effect of caffeine upon cell survival after UV irradiation, it cannot explain the similar effect after MNNG treatment.     

Characterization of chemically induced mutations in the ad-I locus of Schizosaccharomyces pombe

An attempt to assess the frequencies of mutations of the base-pair substitution type and of the addition/deletion type was undertaken in 64 ICR-170-, 28 MNNG- and 50 EMS-induced ad-1 mutant strains of Schizosaccharomyces pombe.By using temperature sensitivity, osmotic remediability, and interallelic complementation, sensitivity to nonsense suppressors and revertibility tests with 2-methoxy- 6-chloro-9-[3-(ethyl-2-chloroethyl)aminopropylamino]acridine dihydrochloride (ICR-170) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) as diagnostic criteria to distinguish between the two types of alterations, the following conclusions were reached: (1) The mutational alteration in all of the MNNG-induced and in at least 74% of the ethyl methanesulfonate(EMS)-induced mutant strains is of the base-pair substitution type; (2) Both types of mutation were found amongst ICR-170-induced strains.     

Induction of gene conversion in yeast cells continuously cultured at high radiation background

The induction of genetic damage was investigated by culturing diploid yeastSaccharomyces cerevisiae D7 cells continuously at radiation levels ranging from 0.383 µSv/h to 1.275 mSv/h by selecting appropriate concentrations of tritiated water in the growth medium. These radiation levels correspond to 3–10000 times the natural background. Parameters such as growth kinetics, gene conversion frequency at background radiation and after a challenging dose of acute gamma-radiation or alkylating agentN-methyl-N-nitro-N-nitrosoguanidine (MNNG) were assessed. The gene conversion frequency in most of the assays was in the range of 5–10 convertants per 10⁶ cells, as in the case of controls. However, a number of the cultures showed conversion frequencies above 20 per 106 viable cells. This stochastic phenomenon occurred more frequently in cells which were incubated at higher radiation levels and for longer durations. This suggests that radiation is responsible for the phenomenon. When subculturing continued beyond 900 h, gene conversion frequencies reverted back to normal values in all cultures in spite of elevated background radiation levels, thus suggesting an adaptive response. The generation time of the cells was 78 min in all cultures irrespective of the radiation level. The response of the cells cultured at elevated background radiation levels to subsequent challenging treatment with gamma-radiation or MNNG was identical to that of the control cultures. Our results suggest that in eukaryotic yeast, low-level radiation may induce an adaptive response to chronic radiation, whereas no such response could be detected when the cells were challenged with acute high-dose exposure or with MNNG.     

Correlation between lethality and DNA single-strand breaks in Bacillus subtilis cells treated with N-methyl-N'-nitro-N-nitrosoguanidine

The effects of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) treatment two Bacillus subtilis strains which exhibit different UV sensitivities were monitored at both the cellular (survival) and subcellular (DNA strand break) levels. The MNNG-induced single strand DNA breaks (SSB) in either strain, as measured by alkaline sucrose gradient centrifugation, were shown to be well correlated with lethality. These DNA lesions were shown by a computer simulation to be randomly induced. Cell survival after MNNG treatment is inversely related to the number of replication forks per cell which in turn depends upon the doubling time of the culture and the growth phase. The production of single-strand breaks and cell killing are proportional to the log of the initial MNNG concentration and may imply that decomposition of the mutagen is (pseudo) second order.     

Induction of 6-thioguanine resistance in synchronized human fibroblast cells treated with methyl methanesulfonate, N-acetoxy-2-acetylaminofluorene and N-ethyl-N′-nitro-N-nitrosoguanidine

Chemical induction of 6-thioguanine resistance was studied in synchronized human fibroblast cells. Cells initially grown in a medium lacking arginine and glutamine for 24 h ceased DNA synthesis and failed to enter the S phase. After introduction of complete medium, the cells progressed to the S phase after 16 h. DNA synthesis peaked 20 h after removal of nutrient stress and declined.Mutations were induced in S-phase cells by methyl methanesulfonate (MMS), N-acetoxy-2-acetylaminofluorene (NA-AAF) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Chemical treatments resulted in an increase in the absolute number of mutant colonies and in a dose-dependent mutation frequency. In this report, we show that NA-AAF evokes a temporal pattern of mutation in synchronized cells, with such mutations being induced only during the S phase. Evidence indicates that presence of S-phase cells in the treated cultures is a prerequisite for the induction of mutations.     

Properties of a UV-sensitive Neurospora strain defective in pyrimidine dimer excision

The UV-sensitive Neurospora strain uvs-2 is known to resemble the excision-defective uvr mutants of E. coli K12 in being both excision-defective and highly UV mutable. As shown in this report, the uvs-2 strain also resembles the uvr mutants in its ability to remain photoreactivable when held in the dark for 2 h between UV-irradiation and photoreactivating light exposure, and in its maintenance of the same spontaneous deletion rate as wild type strains.Unlike the E. coli uvr mutants, however, this strain is sensitive to ionizing radiation and shows an increase in survival when held for 2 h in distilled water before plating (liquid-holding recovery [LHR]). The strain is three times more sensitive to X-rays than the wild type strain. It is also sensitive to nitrosoguanidine (MNNG). Sensitivity to UV, X-rays and MNNG appears to be under the control of a single gene.These properties suggest that the repair defect in the Neurospora uvs-2 mutant is different from those of the uvr mutants of E. coli K12.     

Genotoxic effects of chemicals in the single cell gel (SCG) test with human blood cells in relation to the induction of sister-chromatid exchanges (SCE)

In a comparative study, henzo[a]pyrene (BaP), cyclophosphamide (CP), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and tetrachloroethylene (PER) were tested for their ability to induce genotoxic effects in the single cell gel (SCG) test and the sister-chromatid exchange (SCE) test with human blood cells. MNNG as well as S9 mix activated BaP- and CP-induced DNA effects in both tests in a dose-dependent manner. While the range of concentrations which induced DNA migration or SCE was the same for MNNG and for Bap, much higher CP concentrations were necessary for a positive response in the SCG test than in the SCE test. PER was tested in the absence and in the presence of S9 mix and neither induced DNA migration nor increased SCE frequencies. In these experiments, a clear cytotoxic effect of PER was observed. To investigate a possible influence of DNA repair on the effects in the SCG test, cells were treated for 2 h and further incubated for 1 h after removal of the test substance. This procedure caused a clear decrease in induced DNA migration in experiments with Bap and CP, whereas no reduction was found with MNNG. This modified protocol did not lead to the detection of DNA effects after treatment with PER. The results indicate that the SCG test responds to various DNA lesions and does not seem to be sensitive to non-genotoxic cell killing. Its sensitivity obviously depends on the type(s) of induced DNA lesions and the effects can be modified by DNA repair processes in a complex manner. For the detection of genotoxic properties of chemicals with the in vitro SCG test, a single evaluation at the end of the exposure period seems to be sufficient.     

Increased sensitivity of xeroderma pigmentosum cells to some chemical carcinogens and mutagens

The clone-forming capacity and level of DNA repair was examined on normal human cells and repair-deficient Xeroderma pigmentosum (XP) fibroblasts exposed to various chemical carcinogens and mutagens.The cultured fibroblasts were treated for 90 min with the carcinogenic and mutagenic 4-nitroquinoline 1-oxide (4NQO), 4-hydroxyaminoquinoline 1-oxide (4HAQO), 2-methyl-4-nitroquinoline 1-oxide (2-Me-4NQO), 3-methyl-4-nitropyridine 1-oxide 3-Me-4NPO) and the non-carcinogenic 6-nitroquinoline 1-oxide (6NQO). The response of the cells to the N-oxides was compared to that induced by the mutagen and carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and UV-irradiation.The XP cells showed (1) a reduced level of DNA repair synthesis when exposed to various carcinogenic N-oxides, (2) no unscheduled DNA synthesis following 6NQO and (3) a normal degree of DNA repair synthesis after treatment with MNNG.When the clone-forming capacity was examined the XP cells exhibited (1) a higher increased sensitivity to the various carcinogenic N-oxides, (2) no reduction in the clone formation following 6NQO and (3) a sensitivity virtually comparable to that of normal cells after treatment with MNNG.The results suggest a link between extent of DNA damage, level of DNA repair and degree of sensitivity in human cells exposed to various chemical carcinogens and which induce DNA alterations that cannot be repaired by DNA repair synthesis.     

Unique acceptors for poly(ADP-ribose) in resting,proliferating and DNA-damaged human lymphocytes

Acceptor proteins for poly(adenosine diphosphoribosyl)ation were determined in resting human lymphocytes, in lymphocytes with N-methyl-N′-nitro-N-nitrosoguanidine-induced DNA damage and in lymphocytes stimulated to proliferate by phytohemagglutinin. Kinetic studies showed that the increase in ADP-ribosylation which occurred in response to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) treatment was greater in magnitude but more transient in duration than that which occurred in phytohemagglutinin-stimulated cells. Gel electrophoretic analyses revealed that MNNG treatment and phytohemagglutinin stimulation both caused an increase in ADP-ribosylation of poly(ADP-ribose) polymerase and core histones. In MNNG-treated cells, an increase in ADP-ribosylation of histone H1 was also observed. In contrast, phytohemagglutinin-stimulated cells showed no increase in ADP-ribosylation of histone H1. In MNNG-treated cells there was also ADP-ribosylation of a protein of molecular weight 62 000, while in phytohemagglutinin-stimulated cells there was a marked increase in ADP-ribosylation of a protein of molecular weight 96000. MNNG treatment of phytohemagglutinin-stimulated cells produced a pattern of ADP-ribosylation that appeared to be due to the combined effects of the individual treatments. 3-Aminobenzamide effectively inhibited ADP-ribosylation under all treatment conditions.     

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.     

Lack of correlation between DNA-methylating activity and appearance of the immunogenic phenotype in clones of a murine lymphoma treated with mutagens

Summary The relationship between induction of novel immunogenicity by xenogenizing chemicals and DNA-methylating activity in murine tumors was investigated at the clonal level in L1210Ha cells treated with 5-azacytidine, N-methyl-N-nitro-N-nitrosoguanidine (MNNG) or 1-(p-chlorophenyl)-3,3-dimethyltriazene (DM-Cl). Cells were exposed to the drugs in vitro, cloned by limiting dilution, and assayed for transplantation immunogenicity and 5-methylcytosine content. The results showed that 0% (0/29, 5-azacytidine), 6.8% (2/29, MNNG) and 87.5% (28/32, DM-Cl) of the resulting clones were highly immunogenic, as judged by their tumorigenicity in intact compared to immunodepressed hosts. Frequency distribution analysis of the 5-methylcytosine content of drug-treated and parental clones showed that the methylation pattern was not significantly modified by tumor exposure to either 5-azacytidine or MNNG, and the two immunogenic clones induced by MNNG had methylcytosine levels very close to the 50th percentile value. In contrast, the extent of DNA methylation was increased in the cells treated with DM-Cl, but no obvious association was found between methylation status and immunogenicity of the drug-treated clones. In four 5-azacytidine-treated clones that displayed little or no immunogenicity, additional rounds of drug exposure led to progressive DNA demethylation, but failed, as a rule, to enhance tumor cell immunogenicity. Taken together, the present data indicate that, at least for the examined tumor, immunogenic variants are generated by mutagen treatment at high (MNNG) or very high (DM-Cl) frequencies under conditions in which hypomethylation-induced antigen amplification is unlikely.This work was supported by Progetto Finalizzato Oncologia, C. N. R, Rome-Italy, grant no. 87.01423.44 Abbreviations used: MNNG, N-methyl-N-nitro-N-nitrosoguanidine; DM-Cl, 1-(p-chlorophenyl)-3,3-dimethyltriazene; MST, difference (days) between median survival times of intact and irradiated mice injected with the same cells.     

Modification of three enzymes of the β-ketoadipate pathway by N-methyl-N′-nitro-N-mtrosoguanidine

The sensitivities of three enzymes of the β-ketoadipate pathway to inactivation by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) were determined in vivo and in vitro under conditions compatible with mutagenesis.One enzyme, β-ketoadipate enol-lactone hydrolase, is very sensitive to inactivation by low concentrations of MNNG. This enzyme is also sensitive to inactivation by N-ethylmaleimide and mercurial reagents. The free sulfhydryl content of native enol-lactone hydrolase was determined to be two moles free sulfhydryl per mole of enzyme. A 95% inactivation of enol-lactone hydrolase by MNNG results in a masking of slightly more than one mole sulfhydryl per mole enzyme.Muconate lactonizing enzyme is moderately sensitive to inactivation by low concentrations of MNNG, but is not inactivated by sulfhydryl reagents. Muconolactone isomerase is resistant to inactivation by low concentrations of MNNG and is not inactivated by sulfhydryl reagents. Upon exposure to high concentrations of MNNG, muconolactone isomerase is rapidly inactivated. Spectrophotometric evidence indicates the lysine residues are nitroguanidinated proportionally with a loss in the enzymatic activity.These data indicate that the exposure of cells to low concentrations of MNNG should affect the activity of enzymes with essential sulfhydryl groups.     

Chromosomal aberration,mutation and morphological transformation of Syrian hamster embryonic cells after exposure to methylnitrosocyanamide

Hamster embryonic fibroblasts were treated directly with various concentrations of methylnitrosocyanamide (MNC), a nitrosated product of methylguanidine (MG) or N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Then they were examined for chromosomal aberrations, morphological transformation and mutations resistant to 8-azaguanine (8AG) and 6-thioguanine (6TG). Direct treatment with 2 to 10 × 10^?6 M MNC caused a marked, dose-dependent appearance of 8AG- and 6TG-resistant mutations. The ability of MNC to induce mutations was similar to that of MNNG. Cultured embryonic fibroblasts in metaphase plates also showed a marked dose-dependent increase in chromosomal aberrations within 24 h after direct treatment with MNC of MNNG. Moreover, MNC and MNNG caused similar rates of morphological transformation.     

Appearance of Virulent Bacteriophages in Lysogenic E. coli Cultures after Prolonged Growth in the Presence of Triethylenemelamine

Continuous culture of coli 12λ, P22, 600-434, 600-21, and 600-299 in the presence of triethylenemelamine (TEM) results in the appearance of a new virulent virus which attacks the parent culture. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) is effective with 600-21 and ultraviolet light with 12λ and 600-21. The cultures which produce the virulent virus continue to do so indefinitely in the absence of the mutagen, but are not lysogenic for the virus. Most of the cells in such cultures are resistant to the virus and do not produce any, but there are a few mutant cells sensitive to the virus and the virus multiplies by infection of these sensitive mutants.     

Non-phenotypic selection of N-methyl-N′-nitro-N-nitrosoguanidine-directed mutation at a predicted hotspot site

The striking mutational specificity of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) exhibited in the lacI gene in Escherichia coli allows comment on the phenotypic consequences of mutation at specific sequences that are not recovered after MNNG mutagenesis. We predict that the I⁺ phenotype is maintained when such silent positions are substituted by amino acids whose codons are generated by the MNNG-directed G:C → A:T transition. We chose the mutationally silent Gly200 codon (an MNNG hotspot motif sequence) to test this prediction. Through MNNG mutagenesis we have generated, identified and isolated a G:C → A:T transition at position 627 (5′-GG-C3′) under non-selective conditions which creates the Gly200→Asp substitution. The I⁺ phenotype is retained for this altered repressor.