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)     

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.     

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.     

Evaluation of drug candidates in a battery of short-term genetic toxicology assays: overview

2-Hydroxy-3-methoxybenzaldehyde (omicron-vanillin), the antimutagenic effect of which has been reported on mutagenesis induced by 4-nitroquinoline 1-oxide (4NQO) in Escherichia coli WP2s, enhanced N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutagenesis in the same strain. A remarkable enhancement of mutagenesis provoked by N-methyl-N-nitrosourea (MNU) was also observed by the addition of omicron-vanillin. No enhancing effect was observed on mutagenesis induced by other mutagens such as methyl methanesulfonate (MMS), dimethylsulfate, N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), N-ethyl-N-nitrosourea (ENU), ethyl methanesulfonate, diethylsulfate, 4NQO and furylfuramide (AF-2). On the contrary, omicron-vanillin greatly suppressed AF-2- and 4NQO-induced mutagenesis and showed a slight suppressing effect against mutagenesis induced by MMS, ENNG and ENU. One possible explanation for the enhancing effect of omicron-vanillin on the mutagenesis induced by MNNG or MNU in E. coli WP2s may be inhibition of an inducible adaptive response. Among 7 derivatives of omicron-vanillin, 2-hydroxy-3-ethoxy-benzaldehyde, omicron-hydroxybenzaldehyde and m-methoxybenzaldehyde showed an enhancing effect on MNNG-induced mutagenesis.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Micronucleated reticulocyte induction by ethylating agents in mice.

Six model ethylating agents were tested for clastogenic potency by means of a new technique of the micronucleus assay with mouse peripheral blood cells using acridine orange (AO)-coated slides, to evaluate the test. The alkylating agents were: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), N-ethyl-N-nitrosourea (ENU), diethylsulfate (DES), ethyl methanesulfonate (EMS), epichlorohydrin (ECH) and ethylene dibromide (EDB). The animals were given a single intraperitoneal injection of the following doses of the chemicals: ENNG and ENU, 25, 50 and 100 mg/kg; EMS and DES, 100, 200 and 400 mg/kg body weight. For EDB and ECH, the doses were 50, 100 and 200 mg/kg, given twice, 24 h apart. Before and after the injection, blood samples were taken from the tails at 24-h intervals up to 72 h and preparations were made on AO-coated slides. For each dose group, 4 animals were used and 1000 reticulocytes were examined per slide for the presence of micronuclei. At the optimum induction time of 48 h, ENU induced micronucleated reticulocytes (MNRETs) at all 3 doses. ENNG and EMS induced MNRETs significantly at 2 dose levels each and DES only at the highest dose. ECH and EDB failed to induce MNRETs. On the basis of the dose of chemical needed to double the spontaneous frequency, the order of clastogenic potency was ENU greater than ENNG greater than EMS greater than DES. The results obtained compared favorably with those from other in vivo methods. The present technique proves to be simple, flexible and relatively sensitive. Shifts in the optimum induction peak in individual animals and by some chemicals can be picked up easily which is important when testing weak mutagens and chemicals with an unknown mechanism of action.     

A study of the growth inhibition of Lactobacillus casei by N-alkyl homologs of N-nitro-N-nitrosoguanidine.

N-methyl-(MNNG), N-ethyl-(ENNG), and N-propyl-(PNNG) derivatives of N'-nitro-N-nitrosoguanidine inhibited the growth of Lactobacillus casei in the order of potency, MNNG greater than ENNG greater than PNNG. L-Cysteine, gluathione, and dithioerythritol reversed the inhibition on a molar basis. The -SH compounds accelerated the loss of the N-nitroso group in vitro, yielding non-inhibitory N-alkyl nitroguanidines. The significance of the loss of the nitroso group and the size of the N-alkyl group is discussed.     

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.     

Isolation of a mutant of Salmonella typhimurium strain TA1535 with decreased levels of glutathione (GSH-). Primary characterization and chemical mutagenesis studies

A mutant of Salmonella typhimurium strain TA1535 with decreased glutathione (GSH) levels was isolated after treatment with UV and selection for N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) resistance; this GSH- mutant also exhibited increased resistance to MNNG, the methyl analog of ENNG. Estimation of the cellular GSH content showed that the GSH- derivative contained about 20% of the GSH levels found in TA1535. In mutagenicity tests (hisG46 leads to His+), the GSH- strain required the presence of GSH or L-cysteine in the medium for an optimal phenotypic expression and/or growth of spontaneous and induced His+ revertants, and may, therefore, be allelic to cys mutants of Salmonella described earlier. The mutagenic activity of MNNG, ENNG and 1,2-dibromoethane (DBE), but not that of N-ethylnitrosourea (ENU), was strongly reduced in TA1535/GSH-; pretreatment of the strain with GSH restored the mutagenicity of the first 3 chemicals to levels normally found in TA1535. The results support the current view that MNNG, ENNG and DBE, but not ENU, can be activated via reaction with GSH to species of higher reactivity and mutagenicity. It is concluded that the present GSH- strain can be used to study more systematically the role of GSH in the bioactivation and -deactivation of xenobiotics to mutagenic factors.     

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.     

Sister-chromatid exchanges (SCEs), cell survival and mutation in HeLa s3 cells with different sensitivity to alkylating agents; evidence that SCE induction and cell survival or mutation induction are dissociable

We previously isolated N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-resistant cells, MR from HeLa S3 Mer- cells. In the present study, we have isolated 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU)-resistant cells, ACr. The MR cells had only a little O6-methylguanine-DNA methyltransferase (MT) activity, while the ACr cells had increased MT activity and also became resistant to the cytotoxic effect of MNNG. We compared the induction of sister-chromatid exchanges (SCEs), cell survival and mutation in these HeLa S3 cells with different sensitivity to MNNG. The ACr cells were much more resistant than the parental HeLa S3 Mer- cells to cytotoxicity, mutagenicity and SCE induction by MNNG, showing a positive correlation between SCE induction and cell killing or mutation. In contrast, this positive relationship was not observed between HeLa S3 Mer- and MR cells. These results suggest that O6-methylguanine (O6-MeG) is involved in the induction of the biological effects of MNNG such as cytotoxicity, mutagenicity and SCEs, and also indicate that SCE induction does not always correlate with cell killing and mutation.