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.     

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.     

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.     

In vitro developmental toxicity of five direct-acting alkylating agents in rodent embryos: structure-activity patterns

Five direct-acting alkylating agents were examined qualitatively and quantitatively for their ability to produce developmental toxicity in rodent postimplantation embryos. These agents were structurally related and were capable of donating either a methyl (methylnitrosourea, MNU; methylnitronitrosoguanidine, MNNG; methyl methanesulfonate, MMS) or ethyl (ethylnitrosourea, ENU; ethyl methanesulfonate, EMS) group to nucleophiles. These agents' reactivities were known to differ. In day 10 rat embryos in vitro a single, 2-hour exposure was shown to be sufficient to elicit dose-dependent increases in embryo lethality and malformations. Qualitatively, the patterns of embryo malformations reported in treated embryos paralleled those observed in in vivo studies, especially in regard to adverse effects on central nervous system and craniofacial systems. Quantitatively, the order of potency of these agents in vitro was: MNNG greater than MNU greater than ENU greater than MMS greater than EMS. In vivo studies reported a different order of potency. In vitro, methylating agents were consistently more potent than ethylating agents. Other chemical properties such as nucleophilic reactivity or half-life under physiological conditions could not explain observed potency relationships. Future investigation of other chemical properties of these agents such as specific alkylation and carbamylation reactivities may expand these initial structure-activity observations.     

Cytotoxicity and mutagenicity of alkylating agents in cultured mammalian cells (CHO/HGPRT system): mutagen treatment in the presence or absence of serum

Cytotoxicity and mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells (CHO/HGPRT system) were measured for a range of concentrations of 6 alkylating agents [methyl and ethyl methanesulfonate (MMS, EMS), N-methyl- and N-ethyl-N'-nitro-N-nitrosoguanidine (MNNG, ENNG), and methyl- and ethyl-nitrosourea (MNU, ENU)] to determine the effect of the presence or absence of serum during the time of mutagen treatment. Cultures were treated with the mutagens for 5 h, a time period which results in no growth inhibition in the absence of serum, to estimate the potential decrease in effective mutagen dose to the cells which might result from reactivity with the serum proteins. With all 6 agents, identical results were found for cytotoxicity and for mutagenicity regardless of the presence or absence of serum during treatment. This finding demonstrates that the use of serum in cell-culture medium does not present any problems in apparent dosimetry studies, at least with these alkylating agents.     

Enhanced survival and reduced mutation and aberration frequencies induced in 79 Chinese hamster cells pre-exposed to low levels of methylating agents

Exposure of V79 Chinese hamster cells to a single very low (sub-toxic and sub-clastogenic) dose of MNU or MNNG made these cells resistant to the toxic, mutagenic and clastogenic activities of the same agents given 6 h later. Cell survival was increased nearly 2-fold under optimal conditions when compared with the non-pretreated controls. Aberration frequencies were reduced to nearly half the control values (cells not pretreated). This was observed for a wide range of pretreatment concentrations and at different recovery times. The effect of mutagen pretreatment was most pronounced with respect to the induction of TG resistance, which became drastically reduced. The data indicate the existence of an adaptive repair pathway in V79 cells which may be induced by very low levels of methylating agents and which is error-free in handling lesions responsible, at least partially, for reproductive cell death, mutations and chromosomal aberrations.     

The influence of the nucleotide excision-repair system on mutagenesis in Salmonella typhimurium LT2 after exposure to low doses of monofunctional alkylating agents.

The role of nucleotide excision repair in the mutagenicity of the monofunctional alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), and N-ethyl-N-nitrosourea (ENU) in Salmonella typhimurium was examined. The mutagenic potential of the mutagenic agents used increased in the following order: MMS less than ENU less than ENNG less than MNNG. The results obtained confirm the involvement of nucleotide excision repair in the removal of mutagenic lesions from the DNA of S. typhimurium cells exposed to high doses of methylating as well as ethylating agents. At the low doses of all the alkylating agents used, the nucleotide excision repair-proficient strain was mutagenized more efficiently than the uvrB mutant. This phenomenon, a consequence of competition between nucleotide excision-repair enzymes and constitutive O6-methylguanine-DNA methyltransferase, is discussed.     

Prophage induction in Haemophilus influenzae and its relationship to mutation by chemical and physical agents

It is known that UV, X-rays, MMC and MMS are not mutagenic for H. influenzae, whereas HZ, EMS and MNNG are potent mutagens for this bacterium. All of these agents, however, are known to be both mutagenic and able to induce prophage in E. coli. We report here that all the agents except HZ induce prophage in H. influenzae, and EMS even induces in the recombination-defective recl mutant, which is non-inducible by UV, MMC, MNNG and MMS. MMS did not cause single-strand breaks or gaps in DNA synthesized after treatment of H. influenzae, but EMS and MNNG produced them. EMS caused more breaks in DNA synthesized before treatment than in that synthesized after treatment. On the other hand we did observe such breaks or gaps induced in E. coli in DNA synthesized posttreatment by EMS as well as by MMS and MNNG, at comparable survival levels.     

Cell killing by various monofunctional alkylating agents in Chinese hamster ovary cells

Cell killing by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), and methyl methanesulfonate (MMS) was measured in Chinese hamster ovary (CHO) cells using the colony-formation assay. Cell killing by these agents was determined in exponentially growing asynchronous cells, in synchronous cells as a function of cell-cycle position and in nondividing cells. Distinct differences in the cytotoxic effect of the 4 alkylating agents were found in respect to dose-response, cell cycle phase-sensitivity and growth state. MNNG and MNU showed the same biphasic dose-survival relationship in exponentially growing cells, with an initial steep decline followed by a shallow component. The shallow component disappeared in growth-arrested cells. MNNG and MNU differed, however, in the cell-cycle age response. No cell-cycle phase difference was seen with MNNG, whereas cells in G1 seemed more sensitive to MNU than cells in S phase. MMS and ENU both showed shouldered dose-response curves for exponentially growing asynchronous cells, and the same cell-cycle pattern for synchronous cultures with cells in early S phase being the most sensitive. However, survival of nondividing cells versus dividing cells was reduced much more by MMS than by ENU. Caffeine, which interferes with the regulation of DNA synthesis and is known to modify cell killing by DNA-damaging agents, enhanced cell killing by all agents. It is concluded that there must be a number of factors which contribute to cell killing by monofunctional alkylating agents, and that besides alkylation of DNA reaction with other cellular macromolecules should be considered.     

Inducible DNA-repair systems in yeast: competition for lesions

DNA lesions may be recognized and repaired by more than one DNA-repair process. If two repair systems with different error frequencies have overlapping lesion specificity and one or both is inducible, the resulting variable competition for the lesions can change the biological consequences of these lesions. This concept was demonstrated by observing mutation in yeast cells (Saccharomyces cerevisiae) exposed to combinations of mutagens under conditions which influenced the induction of error-free recombinational repair or error-prone repair. Total mutation frequency was reduced in a manner proportional to the dose of 60Co-gamma- or 254 nm UV radiation delivered prior to or subsequent to an MNNG exposure. Suppression was greater per unit radiation dose in cells gamma-irradiated in O2 as compared to N2. A rad3 (excision-repair) mutant gave results similar to wild-type but mutation in a rad52 (rec-) mutant exposed to MNNG was not suppressed by radiation. Protein-synthesis inhibition with heat shock or cycloheximide indicated that it was the mutation due to MNNG and not that due to radiation which had changed. These results indicate that MNNG lesions are recognized by both the recombinational repair system and the inducible error-prone system, but that gamma-radiation induction of error-free recombinational repair resulted in increased competition for the lesions, thereby reducing mutation. Similarly, gamma-radiation exposure resulted in a radiation dose-dependent reduction in mutation due to MNU, EMS, ENU and 8-MOP + UVA, but no reduction in mutation due to MMS. These results suggest that the number of mutational MMS lesions recognizable by the recombinational repair system must be very small relative to those produced by the other agents. MNNG induction of the inducible error-prone systems however, did not alter mutation frequencies due to ENU or MMS exposure but, in contrast to radiation, increased the mutagenic effectiveness of EMS. These experiments demonstrate that in this lower eukaryote, mutagen exposure does not necessarily result in a fixed risk of mutation, but that the risk can be markedly influenced by a variety of external stimuli including heat shock or exposure to other mutagens.     

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)     

Preventive action of thioethers towards in vitro DNA binding and mutagenesis in E. coli K12 by alkylating agents

Thioethers are effective scavengers of electrophilic metabolites derived from the hepatocarcinogen N-hydroxy-2-acetylaminofluorene (van den Goorbergh et al., 1987). In this study 2 of these thioethers, 4-(methylthio)benzoic acid (MTB) and its methylester, methyl 4-(methylthio)benzoate (MMTB), have been tested for their ability to prevent in vitro DNA binding and mutation induction in E. coli K12 by the direct alkylating agents ethylnitrosourea (ENU), methylnitrosourea (MNU), ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS). In addition to MTB and MMTB, the thioether L-methionine (Met), and the thiols glutathione (GSH) and L-cysteine (Cys) were included for reasons of comparison. MTB was able to (partially) prevent DNA binding and mutation induction by ENU. However, this thioether was ineffective with EMS. DNA binding and mutagenesis by EMS were (partially) prevented by GSH and Cys, while these thiols could not prevent DNA binding and mutation induction by ENU. MMTB was unable to prevent mutation induction by these ethylating agents. With the methylating agents, similar effects of MTB were observed: MTB effectively prevented mutation induction by MNU while it was much less effective towards MMS. GSH and Cys were comparably effective as antimutagenic agents towards both methylating agents. Met was unable to prevent either DNA binding or mutation induction by these agents. Taken together, the results show that aromatic thioethers are able to trap genotoxic electrophiles derived from the nitrosoureas ENU and MNU, and may therefore act as potential anticarcinogens towards these agents, which are only poorly detoxified by GSH.     

Dependency of the yield of sister-chromatid exchanges induced by alkylating agents on fixation time. Possible involvement of secondary lesions in sister-chromatid exchange induction

Chinese hamster V79 cells were pulse-treated (for 60 min) with various mutagens three, two or one cell cycles before fixation (treatment variants A, B and C, respectively) and the frequencies of induced SCEs were analysed and compared. The degree of increase in frequency of SCEs with dose in the treatment variants depended on the mutagen used. For the methylating agents MNU, MNNG and DMPNU, high yields of SCEs were obtained in the treatment variants A and B, and there was no difference in the efficiency with which these agents induced SCEs in these treatment variants. In the treatment variant C, however, no SCEs were induced with mutagen doses yielding a linear increase in SCE frequency in treatment variants A and B. A slight increase in SCE frequency in treatment variant C was observed only when relatively high doses of MNU or MNNG were applied. Like the above agents, EMS, ENU and MMS induced more SCEs in treatment variants A and B than in C, but for these agents treatment variant B was most effective and SCEs were induced over the entire dose range, also in treatment variant C. As opposed to the methylating and ethylating agents, MMC induced SCEs with high efficiency when treatment occurred one or two generations prior to fixation. There was no difference in SCE frequency between these treatment variants. MMC was completely ineffective for the induction of SCEs when treatment occurred three generations before fixation. The unexpectedly low SCE frequencies induced by the methylating and ethylating agents when treatment occurred one generation before fixation were not due to the exposure of cells to BrdU prior to mutagen treatment. From the results obtained, it is concluded that DNA methylation and ethylation lesions give rise to SCEs only with very low probability during the replication cycle after the lesion's induction, and that subsequent lesions produced during or after replication of the methylated or ethylated template (secondary lesions) are of prime importance for SCE formation after alkylation. For MMC, however, primary lesions seem to be most important for SCE induction.     

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.     

Base alterations in yeast induced by alkylating agents with differing Swain-Scott substrate constants.

The base alterations induced by four alkylating agents, methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), N-nitroso-N-methylurea (MNU), and N-nitroso-N-ethylurea (ENU), have been determined at the URA3 locus in the yeast Saccharomyces cerevisiae. The mutagen treatment was carried out on yeast cells in the logarithmic phase of growth. The mutants were selected by their resistance to 7.3 mM-5-fluoroorotic acid at pH 3.8. DNA sequence analysis was carried out by the dideoxy chain termination method. The alkylating agents were selected for their widely differing Swain-Scott substrate constants (s values), which are as follows: MMS, s = 0.83; EMS, s = 0.67; MNU, s = 0.42; ENU, s = 0.26. A higher s value is correlated with a higher ratio of 7-alkylguanine to O6-alkylguanine in native DNA in vitro. 125 forward mutations from URA3----ura3 were sequenced with marked differences in the mutational spectra being observed as the s value changed. Five hotspots were recorded for the four alkylating agents. They were all G.C----A.T transition mutations. There was one common hotspot for all of them; there were two additional ones for the two ethylating agents (ENU and EMS) and two different ones for MNU. Four of the five hotspots have the 5'-GG-3' sequence with the 3'-guanine mutated. It was seen that MMS, which has the highest Swain-Scott substrate constant, yielded the widest array of mutational types. As the substrate constants decreased, the types of mutations became more and more restricted to the G.C----A.T transitions and the A.T----T.A transversions. The transitions are consistent with the concept that mutations arise from O6-alkylation of guanine and alkylation of thymine. The transversions are consistent with the notion of N1-alkylation of adenosine or adenylic acid.     

Radiation equivalent of genotoxic chemicals: Validation in cultured mammalian cell lines

Published data on mutations induced by ionizing radiation and 6 monofunctional alkylating agents, namely EMS, MMS, ENNG, MNNG, ENU and MNU, in different cell lines (Chinese hamster ovary, Chinese hamster lung V79, mouse lymphoma L5178 and human cells) were analysed so that radiation-equivalent chemical (REC) values could be calculated.REC values thus obtained for a given alkylating agent with different cell lines fall within a narrow range suggesting its validation in cultured mammalian cell systems including human.     

Enhancement by alkylating agents of chemical carcinogen transformation of hamster cells in culture

When Syrian hamster embryo cells were pretreated with a weak chemical carcinogen, methyl methanesulfonate (MMS) or ethyl methanesulfonate (EMS), or with a physical agent such as X-irradiation prior to being exposed to a potent cancer-producing chemical, transformation (crisscrossing of cells not seen in control) occurred up to nine times more often than when the cells were not pretreated. The degree of enhancement appears independent of carcinogen dose. The transformation frequency associated with the carcinogens benzo(a)pyrene (BP), dimethylbenz(a)anthracene (DMBA), 3-methylcholanthrene (MCA), N-acetoxy-2-acetylaminofluorene (AcAAF), and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was increased. There are similarities in the enhancement produced by pretreatment of hamster cells with X-irradiation and with alkylating agents: with both, maximum enhancement occurred approx. 48 h after treatment and lethality attributable to the pretreatment was 10–20% relative to control. However, enhancement produced by X-irradiation pretreatment was slightly greater than that obtained with MMS. The exact cause of the enhancement in transformation resulting from the interaction of these agents is not yet known, but the enhancement associated with MMS pretreatment cannot be related to partial cell synchronization or disruption in the cell cycle. Hamster cells pretreated with 250 μM of MMS demonstrated no alteration in normal cel DNA synthesis through 48-h post-treatment. Analysis of unscheduled DNA synthesis by autoradiography or by alkaline sucrose gradients indicated that the damaged DNA was rapidly repaired after treatment. Therefore, repair of DNA damage as it is now understood is probably not involved.     

DNA adduct formation in mouse testis by ethylating agents: a comparison with germ-cell mutagenesis

DNA adduct formation in various organs of mice was determined after i.p. injection with the ethylating agents N-ethyl-N-nitrosourea (ENU), ethyl methanesulfonate (EMS), and diethyl sulfate (DES). The potency of the 3 chemicals to react either at the O6 position of guanine or at the N-7 position of guanine was related to their potency to induce mutations in the specific-locus assay of the mouse. ENU, which produces relatively high levels of O-alkylations (O6-ethylguanine), is primarily mutagenic in spermatogonia of the mouse, whereas EMS and DES, which produce relatively high levels of N-alkylations (7-ethylguanine) in DNA, are much more mutagenic in post-meiotic stages of male germ cells. The relationship between exposure to ENU and the dose, determined as O6-ethylguanine per nucleotide in testicular DNA, is non-linear. However, the relationship between dose and mutation induction in spermatogonia by ENU appears to be linear, which is expected if O6-ethylguanine is the major mutagenic lesion. The relatively high mutagenic potency of EMS and DES in the late stages of spermatogenesis is probably due to the accumulation of apurinic sites which generate mutations after fertilization. A comparison of mutation induction by ENU in spermatogonia and mutation induction in cultured mammalian cells indicates that about 10 O6-ethylguanine residues were necessary in the coding region of a gene to generate a mutation.     

Adaptive response to low dose of EMS or MMS in human peripheral blood lymphocytes

Human peripheral blood lymphocytes stimulated in vitro for 6 hr were exposed to a low (conditioning) dose of ethyl methanesulfonate (EMS; 1.5 x 10(-4) M) or methyl methanesulfonate (MMS; 1.5 x 10(-5) M). After 6 hr, the cells were treated with a high (challenging) concentration of the same agent (1.5 x 10(-3) M EMS or 1.5 x 10(-4) M MMS). The cells that received both conditioning and challenging doses became less sensitive to the induction of sister chromatid exchanges (SCEs) than those which did not receive the pretreatment with EMS or MMS. They responded with lower frequencies of SCEs. This suggests that conditioning dose of EMS or MMS has offered the lymphocytes to have decreased SCEs. This led to the realization that pre-exposure of lymphocytes to low dose can cause the induction of repair activity. This is a clear indication of the existence of adaptive response induced by alkylating agents whether it is ethylating or methylating in human lymphocytes in vitro.