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.     

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.     

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.     

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.     

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.     

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.     

Induced mutation spectra at the upp locus in Salmonella typhimurium: response of the target gene in the FU assay to mechanistically different mutagens

A novel forward mutation assay has been developed in Salmonella typhimurium based on resistance to 5-fluorouracil (FU). The mutational target in the FU assay was determined to be the uracil phosphoribosyl transferase (upp) gene. To validate the upp gene as a suitable target for monitoring a variety of induced mutations, the mutational specificity was determined for five mechanistically different mutagens. The mutagens included a polycyclic hydrocarbon (benzo[a]pyrene, B[a]P), SN1 and SN2 alkylating agents (N-nitroso-N-methylurea, MNU, and methyl methanesulfonate, MMS, respectively), a frameshift mutagen (ICR-191), and an oxidative-damaging agent (hydrogen peroxide, H2O2). Induced mutation frequencies were measured in the presence and absence of the plasmid pKM101 (strain FU100 and FU1535, respectively). pKM101 renders FU100 more susceptible to induced mutation by providing error-prone replicative bypass of DNA adducts. B[a]P, MMS, and H2O2 failed to induce the mutant frequency in FU1535, demonstrating the dependence of pKM101 on induced mutations with these agents. ICR-191 and MNU were not dependent on pKM101, and did significantly induce mutations in FU1535. In contrast to FU1535, all agents significantly induced mutations in FU100. Approximately 60 independent mutants were sequenced for each agent that significantly induced the mutant frequency above background. The resulting mutational spectra illustrated predictable molecular fingerprints based on known mutagenic mechanisms for each agent. The predominant mutations observed were G:C to T:A transversions for B[a]P, A:T to T:A and G:C to T:A transversions for MMS, G:C to T:A transversions and A:T frameshifts for H2O2, G:C frameshifts for ICR-191, and G:C to A:T transitions for MNU. It can be concluded that the upp gene in the FU assay is a sensitive and suitable target to monitor a variety of induced mutations in Salmonella.     

Molecular Analysis of Mutations Induced in the Vermilion Gene of Drosophila Melanogaster by Methyl Methanesulfonate

The nature of DNA sequence changes induced by methyl methanesulfonate (MMS) at the vermilion locus of Drosophila melanogaster was determined after exposure of postmeiotic male germ cell stages. MMS is a carcinogen with strong preference for base nitrogen alkylation (s = 0.86). The spectrum of 40 intralocus mutations was dominated by AT----GC transitions (23%), AT----TA transversions (54%) and deletions (14%). The small deletions were preferentially found among mutants isolated in the F1 (8/18), whereas the AT----GC transitions exclusively occurred in the F2 (6/22). The MMS-induced transversions and deletions are presumably caused by N-methyl DNA adducts, which may release apurinic intermediates, known to be a time-related process. Furthermore, MMS produces multilocus deletions, i.e., at least 30% of the F1 mutants analyzed were of this type. A comparison of the mutational spectra of MMS with that produced by ethylnitrosourea (ENU), also in the vermilion locus of Drosophila, reveals major differences: predominantly transition mutations (61% GC----AT and 18% AT----GC) were found in both the F1 and F2 spectrum induced by ENU. It is concluded that the mutational spectrum of MMS is dominated by nitrogen DNA adducts, whereas with ENU DNA sequence changes mainly arose from modified oxygen in DNA.     

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.     

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.     

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.     

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.     

Specific targets of alkylating agents in nuclear proteins of cultured hepatocytes

We have established a specific correlation between the carcinogenic potency of a series of alkylating agents, with a mechanism of reaction ranging between Ingold's SN1-SN2 (ENU greater than MNU = MNNG greater than EMS greater than DMS = MMS) (Vogel et al., 1979; Bartsch et al., 1983) and specific target sites in the amino acids of nuclear proteins of cultured hepatocytes. More potent carcinogens, that react predominantly with an Ingold's SN1 mechanism, mainly alkylate the amino group of lysine and the guanido group of arginine. Weaker carcinogens, reacting with a mechanism closely resembling an Ingold's SN2, mainly alkylate the sulfhydryl group of the cysteine and the 3 position of the imidazolic ring of histidine. A compound with an intermediate type of reactivity alkylates, to a comparable extent, all 4 of the above-described positions. Although stable DNA damage brought about by alkylating carcinogens is considered to be the most likely cause of neoplastic transformation, epigenetic modifications may also play an important role in the process, especially because of their extreme stability. We have verified the existence of a linear correlation between the Swain-Scott substrate constant (S) of each compound and the amount of alkylation produced at the specific target sites. This type of correlation could be the basis of a 'short-term' genotoxicity assay in a battery of complementary tests.     

Alkylation of cytosine and 5-hydroxymethyl-cytosine by methyl methanesulphonate and N-methyl-N-nitrosourea: its relevance to mutagenesis.

The reaction of cytosine and 5-hydroxymethyl-cytosine (OHMeCyt) with a variety of monofunctional alkylating agents has been investigated to evaluate further the possible role of cytosine alkylation in mutagenesis and the possibility that the immunity of T-even phages to mutation by methyl methanesulphonate (MMS) was due to the unreactivity of OHMeCyt towards this agent. Both cytosine and OHMeCyt reacted equally well with the methylating agents MMS and N-methyl-N-nitrosourea (MNU) affording 6% and less than 1% respectively of the 3-substituted derivative. No product was isolated following subjection of the bases to reaction with ethyl methane-sulphonate (EMS), N-ethyl-N-nitrosourea (ENU) or iso-propyl methane-sulphonate (iPMS).     

Tumour induction by low molecular weight alkylating agents

Low molecular weight alkylating carcinogens, such as nitroso compounds, alkylate guanine of DNA to 7-alkylguanine, but the amount of this product correlates poorly with tumour induction. Loveless postulated that a minor product of alkylation, O-(6)-alkylguanine, may be responsible for mutagenesis and carcinogenesis. He showed that methyl methanesulphonate (MMS) does not produce O-(6)-methylguanine from deoxyguanosine, and in the present study it failed to induce thymic lymphomas or pulmonary adenomas in inbred Swiss mice. Loveless gave evidence that ethyl methanesulphonate (EMS), methylnitrosourea (MNU) and ethylnitrosourea (ENU) did produce O-(6)-alkylguanine, and all three induced pulmonary adenomas in the present study. It has also been shown that both of the alkylnitrosoureas induced thymic lymphomas but ethyl methanesulphonate did not.     

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

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

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.     

The effect of folate deficiency on the hprt mutational spectrum in Chinese hamster ovary cells treated with monofunctional alkylating agents.

Folic acid deficiency acts synergistically with alkylating agents to increase DNA strand breaks and mutant frequency at the hprt locus in Chinese hamster ovary (CHO) cells. To elucidate the mechanism of this synergy, molecular analyses of hprt mutants were performed. Recently, our laboratory showed that folate deficiency increased the percentage of clones with intragenic deletions after exposure to ethyl methanesulfonate (EMS) but not N-nitroso-N-ethylurea (ENU) compared to clones recovered from folate replete medium. This report describes molecular analyses of the 37 hprt mutant clones obtained that did not contain deletions. Folate deficient cells treated with EMS had a high frequency of G>A transitions at non-CpG sites on the non-transcribed strand, particularly when these bases were flanked on both sides by G:C base pairs. Thirty-three percent of these mutations were in the run of six G's in exon 3. EMS-treated folate replete cells had a slightly (but not significantly) lower percentage of G>A transitions, and the same sequence specificity. Treatment of folate deficient CHO cells with ENU resulted in predominantly T>A transversions and C>T transitions relative to the non-transcribed strand. These findings suggest a model to explain the synergy between folate deficiency and alkylating agents: (1) folate deficiency causes extensive uracil incorporation into DNA; (2) greatly increased utilization of base excision repair to remove uracil and to correct alkylator damage leads to error-prone DNA repair. In the case of EMS, this results in more intragenic deletions and G:C to A:T mutations due to impaired ligation of single-strand breaks generated during base excision repair and a decreased capacity to remove O6-ethylguanine. In the case of ENU additional T>A transversions and C>T transitions are seen, perhaps due to mis-pairing of O2-ethylpyrimidines. Correction of folate deficiency may reduce the frequency of these types of genetic damage during alkylator therapy.     

Survival and mutagenesis of bacteriophage T7 damaged by methyl methanesulfonate and ethyl methanesulfonate

We have examined survival and mutagenesis of bacteriophage T7 after exposure to the alkylating agents methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS). It was found that although both alkylating agents caused increased reversion of specific T7 mutations, EMS caused a higher frequency of reversion than did MMS. Exposure of the host cells to ultraviolet light so as to induce the SOS system resulted in increased survival (Weigle reactivation) of T7 phage damaged with either EMS or MMS. However, after SOS induction of the host we did not detect an accompanying increase in mutation frequency measured as either reversion of specific T7 mutants or by generation of mutations in the T7 gene that codes for phage ligase. Neither mutation frequency nor survival of alkylated phage was affected by the umuD,C mutation in the Escherichia coli host nor by the presence of plasmid pKM101. This may mean that the mode of Weigle reactivation that is detected in T7 is not mutagenic in nature.