Potent induction of the adaptive response by a weak mutagen, methyl iodide, in Escherichia coli

Methyl iodide (MeI), a weakly mutagenic and highly chemoselective chemicals, was tested for its abilities to induced the adaptive and SOS responses in E. coli CSH26/pMCP1000 (alkA′-lacZ′) and CSH26/psK1002 (umuC′-lacZ′). MeI induced the adaptive response effectively but gave a very weak SOS response. Its potent ability in inducing the adaptive response was also demonstrated by adaptation to both the mutagenic and killing effects of N-methyl-N-nitrosourea (MNU) in E. coli WP2 cells. Simultaneous treatment with MeI in a non-growth medium slightly increased the mutagenicity of MNU, probably as a result of depletion of the repair enzyme, O⁶-methylguanine-DNA methyltransferase, which is constitutively present in the cells. As MeI itself proved to be only weakly mutagenic, a small part of the adaptive response which we have observed may involve indirect methylation of the repair enzyme by methyl transfer from MeI-induced O⁶-methylguanine residues in DNA. But the extent of the induced adaptive response seems to be much higher than would be expected from the observed weak mutagenicity of MeI. It is therefore suggested that the mechanism of induction of the adaptive response may involve direct methylation of the O⁶-methylguanine-DNA methyltransferase itself.     

Inhibitory effect of cadmium and mercury ions on induction of the adaptive response in Escherichia coli

Cadmium and mercury ions inhibited the promotion of ada and alkA gene expression in the adaptive process induced by methylating agents such as N-methyl-N-nitrosourea (MNU), methyl methanesulfonate (MMS) and methyl iodide in Escherichia coli. In fact, the induction of O6-methylguanine-DNA methyl-transferase (MGTase) by MNU was suppressed in E. coli in the presence of these metal ions. These ions potentiated mutagenesis induced by methylating agents such as MNU and MMS, but not that induced by ethylating agents, UV irradiation, or N4-aminocytidine. These comutagenic effects were observed in wild-type and umuC36 strains of E. coli but not in the ada-5 strain, which is unable to induce the adaptive response. These results suggest that the comutagenic effects of Cd2+ and Hg2+ are due to inhibition of ada and alkA gene expression promoted by methylated MGTase.     

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.     

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.     

Mutagenesis, by methylating and ethylating agents, in mutH, mutL, mutS, and uvrD mutants of Salmonella typhimurium LT2.

Salmonella typhimurium LT2 mutH, mutL, mutS, and uvrD mutants were especially sensitive to mutagenesis by both the recA+-dependent mutagen methyl methane sulfonate and the recA+-independent mutagen ethyl methane sulfonate, but not to mutagenesis by agents such as 4-nitroquinoline-1-oxide and UV irradiation. Similarly, these mutator strains were very sensitive to mutagenesis by the methylating agents N-methyl-N'-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea. The increased susceptibility to mutagenesis by small alkylating agents due to mutH, mutL, mutS, and uvrD mutations was not accompanied by an increased sensitivity to killing by these agents. Various models are discussed in an effort to explain why strains thought to be deficient in methyl-instructed mismatch repair are sensitive to mutagenesis by methylating and ethylating agents.     

Exchange of individual ribosomal proteins between ribosomes as studied by heavy isotope-transfer experiments

Summary We have described previously an inducible response in Escherichia coli which occurs during growth on low levels of the methylating agent, N-methyl-N-nitro-N-nitrosoguanidine (MNNG), and which enables cells both to survive better and to be less mutated by a subsequent challenge dose of MNNG than control cultures (Samson and Cairns, 1977). We show here that this response is distinct from previously characterised pathways of DNA repair, and particularly from the SOS response, which is another inducible effect resulting from DNA damage. An examination of the cross-reactivity of this response with other mutagens has shown that it is a generalised mechanism affecting alkylation damage to DNA. It cannot, however, be induced by UV or the UV-mimetic mutagen, 4-nitroquinoline 1-oxide, nor act on lesions put into DNA by those mutagens.     

Expression of DNA damage-inducible genes of Escherichia coli upon treatment with methylating, ethylating and propylating agents

Several alkylation-inducible genes have been identified by construction of Mu-d1 (Apr lac) fusions to genes whose expression is increased in response to alkylation treatment, but not UV treatment. We have examined the induction of 4 different alkylation-inducible genes by treatment with a variety of methylating and ethylating agents, and a propylating agent. We have compared the induction of the alkylation-inducible genes with the induction of the sulA gene, which is a component of the SOS response to DNA damage. We find that the Ada-regulated adaptive response genes (ada-alkB, alkA and aidB) are induced primarily in response to methylation treatment. The ada-independent aidC gene is induced upon treatment with agents that alkylate predominantly by SN1 nucleophilic attack. aidC induction occurs only when cells are not aerated during treatment. The SOS response, as indicated by sulA induction, is strongly induced by all types of alkylating agents used.     

Mutagenic frequencies of site-specifically located O6-methylguanine in wild-type Escherichia coli and in a strain deficient in ada-methyltransferase.

The adaptive response of Escherichia coli involves protection of the cells against the toxic and mutagenic consequences of exposure to high doses of a methylating agent by prior exposure to low doses of the agent. Ada protein, a major repair activity for O6-methylguanine, is activated to positively control the adaptive response; O6-methylguanine is one of the major mutagenic lesions produced by methylating agents. We investigated the mutation frequency of wild-type Escherichia coli and strains containing the ada-5 mutation in response to site-specifically synthesized O6-methylguanine under conditions in which the adaptive response was not induced. Site-directed mutagenesis and oligonucleotide self-selection techniques were used to isolate the progeny of M13mp18 DNAs constructed to contain O6-methylguanine at any of eight different positions. The progeny were isolated from E. coli strains isogeneic except for deficiency in Ada-methyltransferase repair, UvrABC excision repair, or both. The resulting O6-methylguanine mutation levels at each position were determined by using differential oligonucleotide hybridization. We found that the wild type had up to a 2.6-fold higher mutation frequency than ada-5 mutants. In addition, the mutation frequency varied with the position of the O6-methylguanine in the DNA in the wild type but not in ada-5 mutants; O6-methylguanine lesions at the 5' ends of runs of consecutive guanines gave the highest mutation frequencies. Determination of the mutation frequency of O6-methylguanine in wild-type and mutS cells showed that mismatch repair can affect O6-methylguanine mutation levels.     

Regulation of expression of the cloned ada gene in Escherichia coli

The ada gene of Escherichia coli K12, the regulatory gene for the adaptive response of bacteria to alkylating agents, was cloned in multicopy plasmids. O6-Methylguanine-DNA methyltransferase and 3-methyladenine-DNA glycosylase II, which are known to be inducible as part of the adaptive response, were produced in ada- cells bearing ada+ plasmids, even without treatment with alkylating agents. When such cells had been treated with methyl methanesulfonate, even higher levels of the enzyme activities were produced. Maxicell experiments revealed that the ada gene codes for a polypeptide with a molecular weight of 38 000. We constructed a hybrid plasmid carrying an ada'-lacZ' fused gene, with the proper control region for ada expression. beta-Galactosidase synthesis from the fused gene was strongly induced only when cells were treated with low doses of methylating agents, but was weakly induced with relatively high doses of ethylating agents. The induction was autogenously regulated by the ada gene product, in a positive manner.     

Analysis of mutagenic activity and ability to induce replication of polyoma DNA sequences by different model metabolites of the carcinogenic tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) requires metabolic activation to express its carcinogenic activity. This activation leads to the formation of methylating and pyridyloxobutylating agents. To determine the possible biological effects mediated by each of these metabolic pathways we have studied the activities of model compounds that are metabolized to either a methylating or pyridyloxobutylating species. Each model compound was evaluated for its mutagenic activity in both prokaryotic and eukaryotic cell systems. The model compounds were also tested for their ability to induce asynchronous replication of viral DNA sequences. We demonstrate here that both the methylating model compound acetoxymethylmethylnitrosamine (AMMN) and the pyridyloxobutylating model compound 4-(acetoxymethyl)-1-(3-pyridyl)-1-butanone (NNKOAc) were mutagenic in strains TA98, TA100, and TA1535 but not TA102. While NNKOAc appeared to be 10 times more potent than AMMN in Salmonella, AMMN was found to be a more potent mutagen in mammalian G12 cells. Both chemicals could induce asynchronous replication of polyoma DNA sequences in rat fibroblast cells carrying an integrated copy of the polyoma virus with AMMN appearing to be more active. Measurement of DNA adduct levels suggest that the damage produced by NNKOAc was at least as active as that produced by AMMN when viewed on a per adduct basis. The possible implications of the biological activities exhibited by methylating and pyridyloxobutylating model compounds to NNK induced carcinogenesis are discussed.     

Biological effect of low doses of alkylating agents in drosophila studies

The low dose (0.05-0.1 mM) influence of alkylating agents on germ cell survival and male fertility, the level of embryonic and postembryonic lethality as well as the sex-linked recessive lethal (SLRL) frequency induced by high alkylating agent doses was studied in Drosophila melanogaster. The pretreatment of adult males with low doses of methyl and ethyl methanesulfonate (MMS and EMS) did not change or even enhanced EMS cytotoxicity and mutagenicity in both mature sperm and premeiotic cells. On the contrary, the low EMS dose pretreatment of larvae protected them against higher mutagen doses increasing male fertility, decreasing embryonic and postembryonic lethality in F1, and leading to three-fold reduction in the SLRL frequency in F2. The adaptive response was dependent on the Drosophila developmental stage exposed to challenge mutagen doses, since the protection was maximal in larvae and practically absent when the high dose was administered to adult males. The adaptive response observed does not seem to be associated with DNA repair, but it is rather due to other protective mechanisms.     

A common mechanism for repair of O6-methylguanine and O6-ethylguanine in DNA

The mutagenic effects of several ethylating and methylating agents were assessed in Encherichia coli strains that are defective in the adaptive response to alkylating agents. These mutants were either deficient in the response or expressed it constitutively. When expressed, the repair pathway removed the major mutagenic lesion produced by either methylating or ethylating agents. This lesion was almost certainly O⁶-alkylguanine produced by alkylation of DNA, and the mechanism for its removal was characterized in vitro. E. coli cells expressing the adaptive response contain relatively large amounts of a protein that transfers the methyl group from O⁶-methylguanine to one of its own cysteine residues (Olsson & Lindahl, 1980). This methyltransferase was shown to act in an analogous fashion on O⁶-ethylguanine. Incubation of ethylated DNA with purified transferase led to disappearance of the O⁶-ethylguanine residues, and S-ethylcysteine was simultaneously generated in the protein. The greater sensitivity of E. coli wild-type to ethylating than methylating agents may be explained by a slower repair of O⁶-ethylguanine than O⁶-methylguanine and also a weaker ability of ethylating agents to induce the adaptive response.     

Adaptive response to alkylating agents in the Drosophila sex-linked recessive lethal assay

The adaptive response to alkylating agents was studied in Drosophila assays under various treatment procedures. Pre-treatment of males as well as treatment of females with low doses of EMS (0.05-0.1 mM) did not affect sex-linked recessive lethal (SLRL) rates induced by high doses of this mutagen (10 mM, various feeding duration) in mature sperm cells. Pre-treatment of males with a low dose of MMS (0.1 mM) enhanced mutagenesis induced by the high dose of EMS (10 mM) at different stages of spermatogenesis, the observed effects exceeding the additive action of both mutagens. On the contrary, larval pre-treatment with the adaptive dose of EMS (0.05 mM) resulted in resistance of their germ cells to higher doses of EMS (1 mM). Specifically, offspring production increased while dominant lethality in F(1) as well SLRL frequency in F(2) was significantly reduced as compared with the effects of larval exposure to the challenge dose. Under the conditions tested, the adaptive response of germ cells to alkylating agents was demonstrated in larvae, but not in adult flies.     

Induction of SOS and adaptive responses by alkylating agents in Escherichia coli mutants deficient in 3-methyladenine-DNA glycosylase activities

The induction of SOS and adaptive responses by alkylating agents was studied in Escherichia coli mutants tagA and alkA deficient in 3-methyladenine-DNA glycosylase activities. The SOS response was measured using an sfiA::lacZ operon fusion. The sfiA operon, in the double mutant tagA alkA, is induced at 5-50-fold lower concentrations of all tested methylating and ethylating compounds, as compared to the wild-type strain. In all cases, the tagA mutation, which inactivates the constitutive and specific 3-alkyladenine-DNA glycosylase I (TagI), sensitizes the strain to the SOS response. The sensitization effect of alkA mutation, which inactivates the inducible 3-alkyladenine-DNA glycosylase II (TagII), is observed under conditions which allow the induction of the adaptive response. We conclude that the persistence of 3-methyladenine and 3-ethyladenine residues in DNA most likely leads to the induction of the SOS functions. In contrast, the adaptive response, evaluated by O6-methylguanine-DNA methyltransferase activity in cell extracts, was not affected by either tagA or alkA mutations. The results suggest that the SOS and adaptive responses use different alkylation products as an inducing "signal". However, adaptation protein TagII inhibits the induction of the SOS response to some extent, due to its action at the level of signal production. Finally, we provide conditions to improve short-term bacterial tests for the detection of genotoxic alkylating agents.     

Effect of the direction of DNA replication on mutagenesis by N-methyl-N''-nitro-N-nitrosoguanidine in adapted cells of Escherichia coli

Summary The methylating agent N-methyl-N-nitro-N-nitrosoguanidine preferentially induces G:C to A:T transitions at DNA base pairs with the G in one particular strand of the cI gene in a lambda prophage, in this case the non-transcribed straind, in Escherichia coli cells in which the adaptive response is induced. The same preference is found for the cI gene inserted in the genome in the inverse orientation, so the differential effect is not caused by the direction of motion of the DNA replicating fork.     

Comparative od adaptive response to gamma-radiation and nickel sulfate treatment in human cells

Increased viability of human cells (line rhabdomyosarcoma) to challenge doses of NiSO4 (10(-5)-10(-3) M) was formed when cells were preirradiated with low doses of gamma-radiation (10-14 cGy). Observed adaptive response was similar to radioadaptive response in human fibroblasts, pretreated with low doses of gamma-radiation and challenge dose of the same mutagen. Pretreatment with low concentration of NiSO4 induced in human fibroblasts increased resistance of DNA to the treatment with challenge doses of gamma-radiation and stimulated DNA repair synthesis after treatment with NiSO4 and 4-nitroquinoline-1-oxide. These data confirm the existence of cross-adaptation in the experiments with NiSO4.