Differential mutagenicity of N-methyl-N-nitrosocarbamate insectides in Escherichia coli strains having different DNA repair capacities.

Four isogenic strains of Escherichia coli with the same auxotrophic marker (arg Fam--namely wild-type, uvrA-, polA- and recA-) were used for testing the lethalities and mutagenicities of 1-naphthyl N-methyl-N-nitrosocarbamate (nitroso-NAC), 3-methylphenyl N-methyl-N-nitrosocarbamate (nitroso-MTMC), and 3,4-dimethylphenyl N-methyl-N-nitrosocarbamate (nitroso-MPMC). The strains recA- and polA- showed a similarly higher sensitivity to killing than wild-type and uvrA- after treatments with each of the three chemicals, whereas the strains wild-type, uvrA-, and polA- were equally mutable by these compounds at equal doses. The strain recA- was hardly mutable by nitroso-NAC, but significant levels of Arg+ mutations were observed after treatments with nitroso-MTMC and nitroso-MPMC. These and previous results suggest that both nitroso-MTMC and nitroso-MPMC are similar in their mutagenicity pattern to N-methyl-N'-nitro-N-nitrosoguanidine whereas nitroso-NAC is similar to methyl methanesulfonate or X-rays, and that the major damage to DNA of the three agents is not excisable by the uvrA+-dependent excision repair, probably methylation in DNA.     

Comparative mutability of the Ames tester strains of Salmonella typhimurium by ultraviolet radiation and by 4-nitroquinoline 1-oxide

A standard method for determining mutant frequencies per survivor was used to study the detailed kinetics of reverse mutations of Ames tester strains of Salmonella typhimurium induced by UV and by 4N1O. After UV irradiation, strain TA1538 was non-mutable, but its plasmid-containing derivative TA98 was mutable, whereas TA1535 was mutable and its plasmid-bearing derivative TA100 was about 10-fold more mutable. After treatment with 4NQO, TA98 was less mutable than TA1538, whereas TA100 was more mutable than TA1535 by a factor of 10–50. TA1537 was slightly less mutable than TA1535 by either UV or 4NQO. The differential mutabilities of these strains are briefly discussed in relation to the “hot spot” base sequences for reversion and the nature of DNA damage caused by UV and 4NQO.     

Natural plasmid pSD89 (Cmr) from Salmonella derby K89, which increases the radiation tolerance of Escherichia coli strain K-12]

Several plasmids with molecular mass of 1.3-9 MDa were found in a clinical isolate of Salmonella derby K89 by electrophoresis in agarose gel. One of these plasmids, designated pSD89 (Cmr), was derived from the K89 strain via transformation of the plasmidless recipient S. derby K82 to chloramphenicol resistance. The plasmid-carrying strain K89 and the K82 strain completely cured of plasmids were equally sensitive to the lethal action of UV light, whereas the plasmid-carrying strain was even more sensitive to ionizing radiation than the plasmidless variant. Nevertheless, transformants carrying only plasmid pSD89 (Cmr) were found to be more resistant to gamma-rays and UV light than the recipient. By using an intermediate host Escherichia coli Z80 (r-m+), plasmid pSD89 (Cmr) was introduced into different E. coli K-12 strains: polA-, recA-, uvrA-, umuC-, and the wild-type strain. A slight increase in radioresistance of E. coli wild-type cells and a significant complementation of a repair defect in recA and polA mutants, but not in uvrA and umuC, were observed.     

An assessment of the importance of error-prone repair and point mutations to forward mutation to L-azetidine-2-carboxylic acid resistance in Escherichia coli

By comparison of E. coli WP2 with CM891 (uvrA- pKM101) we found that pKM101 plasmid and uvrA- mutation considerably enhanced both spontaneous and chemically-induced reversion at the trp locus. However, little or no increase was observed for forward mutation at the A2C locus. Furthermore, mutation frequency decline was considerably greater for trp reversion than for mutation to A2Cr. Thus neither error-prone repair nor point mutation seemed likely to be the major mechanism for forward mutation at the A2C locus. Results for spontaneous mutation of recA-, polA- and gyrA- strains showed that polA- and gyrA- gave good increases in forward mutation but not in reversion. It was inferred that deletion, transposition and/or larger chromosomal effects rather than point mutation were mainly responsible for most forward mutation.     

Repair of near-ultraviolet (365 nm)-induced strand breaks in Escherichia coli DNA. The role of the polA and recA gene products.

The action of near-ultraviolet (UV-365 nm) radiation in cellular inactivation (biological measurements) and induction and repair of DNA strand breaks (physical measurements) were studied in a repair-proficient strain and in polA-, recA-, uvrA-, and polA uvrA-deficient strains of Escherichia coli K-12. The induction of breaks in the polA and polA uvrA strains was linear with dose (4.0 and 3.7 X 10(-5) breaks/2.5 X 10(9) daltons/Jm-2, respectively). However, in the recA-, uvrA-, and repair-proficient strains, there was an initial lag in break induction at low doses and then a linear induction of breaks at higher doses with rates of 4.6, 2.8, and 3.2 X 10(-5) breaks/2.5 X 10(9) daltons/Jm-2, respectively. We interpret these strain differences as indicating simultaneous induction and repair of breaks in polymerase 1 (polA)-proficient strains under the 0 degrees C, M9 buffer irradiation conditions that, for maximum efficiency, require both the polA and recA gene products. Strand-break rejoining also occurred at 30 degrees C in complete growth medium. We propose that at least three (and possibly four) distinct types of pathways can act to reduce the levels of 365-nm radiation-induced strand breaks. A quantitative comparison of the number of breaks remaining with the number of lethal events remaining after repair in complete medium at 30 degrees C showed that between one and three breaks remain per lethal event in the wild-type and recA strains, whereas in the polA strain one order of magnitude more breaks were induced.     

Nickel(II) genotoxicity: potentiation of mutagenesis of simple alkylating agents

Many metals have been shown to alter the function of a wide range of enzyme systems, including those involved in DNA repair and replication. To assess the impact in vivo of such metal actions a "Microtitre" fluctuation assay was used to examine the ability of Ni(II) to act as a comutagen with simple alkylating agents. In E. coli, Ni(II) chloride potentiated the mutagenicity of methyl methanesulfonate (MMS) in polymerase-proficient strains (WP2+ and WP2-), but not in polA- strains (WP6 and WP67) or in lexA- (CM561) or recA- (CM571) strains. The absence of UV excision repair (WP2- and WP67) had little, if any, effect. An extended lag phase was seen at 2-4 h in the polA- strains following treatment with Ni(II) chloride and MMS, but normal growth resumed thereafter. Results suggested that mutations induced by MMS were fixed during log phase growth and that more than 2 h of exposure were necessary for potentiation by Ni(II) to be observed. Thus, the extended lag phase probably cannot explain the lack of potentiation. RecA-dependence of the comutagenic effect was corroborated with S. typhimurium TA1535 and TA100. Only in the pKM101 containing strain, TA100, was potentiation of ethyl methanesulfonate (EMS) and MMS by Ni(II) chloride evident. The mucAB genes carried on pKM101 increase the sensitivity of TA100 to a variety of mutagens, providing there is a functional recA gene product. Taken together, the data suggest that Ni(II) acts indirectly, as a comutagen, in bacterial systems, possibly affecting processes involving recA- and/or polA-dependent function(s).     

Lethal and mutagenic effects of radiation and alkylating agents on two strains of mouse L5178Y cells

In previous studies, the two closely related strains of L5178Y (LY) mouse lymphoma cells, LY-R and LY-S, have been shown to differ in their sensitivity to UV and ionizing radiation. Thus, in comparison to strain LY-R, strain LY-S has been found to be more sensitive to the lethal effects of ionizing radiation, more resistant to the lethal effects of UV radiation, but less mutable at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus by both UV and X-radiation. In the present work, the lethal and mutagenic effects of ethyl methanesulfonate (EMS), methyl nitrosourea (MNU) and UV radiation (254 nm) were compared in the two strains. Mutability at the Na+/K+-ATPase locus as well as the HGPRT locus was determined. As previously reported, we found strain LY-S to be more resistant than strain LY-R to the lethal effects of UV radiation. In contrast, strain LY-S was more sensitive to the cytotoxic effects of the two alkylating agents. In spite of these differences in sensitivity, we found strain LY-S to be less mutable than strain LY-R by all 3 agents at the HGPRT locus. At the Na+/K+-ATPase locus, strain LY-S was also less mutable than strain LY-R by equal concentrations of EMS and UV radiation and by equitoxic concentrations of MNU. However, the difference between the strains was much more pronounced at the HGPRT locus than at the Na+/K+-ATPase locus. We have suggested that the interaction of unrepaired lesions in strain LY-S tends to cause an excess of deletions and multilocus effects, which in turn result in a locus-dependent decrease in the recovery of viable LY-S mutant cells.     

Uvm mutants of Escherichia coli K 12 deficient in UV mutagenesis

Summary Selection for defective reversion induction, after UV treatment of E. coli K 12, yielded uvm mutants. These mutants exhibited highly reduced or no UV mutability for all loci tested although they were moderately and normally mutable by X-rays and EMS, respectively. Uvm mutations confer only a slight sensitivity to killing by UV and X-rays and no clear sensitivity to the lethal effect of HN2, EMS or MMS. Growth and viability of untreated uvm cells were normal. The properties of uvm mutants are discussed in relation to those of other relevant mutant types and to some actual problems of induced mutagenesis.     

Analysis of repair processes by the determination of the induction of cell killing and mutations in two repair-deficient Chinese hamster ovary cell lines

Two UV sensitive DNA-repair-deficient mutants of Chinese hamster ovary cells (43-3B and 27-1) have been characterized. The sensitivity of these mutants to a broad spectrum of DNA-damaging agents: UV254nm, 4-nitroquinoline-1-oxide (4NQO), X-rays, bleomycin, ethylnitrosourea (ENU), ethyl methanesulphonate (EMS), methyl methanesulphonate (MMS) and mitomycin C (MMC) has been determined. Both mutants were not sensitive to X-rays and bleomycin. 43-3B was found to be sensitive to 4NQO, MMC and slightly sensitive to alkylating agents. 27-1 was sensitive only to alkylating agents. The results suggest the existence of two repair pathways for UV-induced cytotoxicity: one pathway which is also used for the removal of 4NQO and MMC adducts and a second pathway which is also used for the removal of alkyl adducts. Parallel to the toxicity, the induction of mutations at the HPRT and Na+/K+-ATPase loci was determined. The increased cytotoxicity to UV, MMC and 4NQO in 43-3B cells and the increased cytotoxicity to UV in 27-1 cells correlated with increased mutability. It was observed that the increase in mutation induction at the HPRT locus was higher than that at the Na+/K+-ATPase locus. As only point mutations give rise to viable mutants at the Na+/K+-ATPase locus the lower mutability at this locus suggests that defective excision repair increases the chance for deletions. Despite an increased cytotoxicity to ENU in 27-1 cells the mutation induction by ENU was the same in 27-1 and wild-type cells at both loci, which suggests that the mutations are mainly induced by directly miscoding adducts (e.g. O-6 alkylguanine), which cannot be removed by CHO cells. As EMS and MMS treatment of 27-1 cells caused an increase in mutation induction at the HPRT locus and a decrease at the Na+/K+-ATPase locus it indicates that these agents induce a substantial fraction of other mutagenic lesions, which can be repaired by wild-type cells. This suggests that O-6 alkylation is not the only mutagenic lesion after treatment with alkylating agents.     

The resistances of Micrococcus radiodurans to killing and mutation by agents which damage DNA

The resistance of Micrococcus radiodurans to the lethal and mutagenic action 3f ultraviolet (UV) light, ionising (γ) radiation, mitomycin C (MTC), nitrous acid (NA), hydroxylamine (HA), N-methyl-N′-nitro-N-nitrosoguanidine (NG), ethylmethanesulphonate (EMS) and β-propiolactone (βPL) has been compared with that of Escherichia coli B/r.M. radiodurans was much more resistant than E. coli B/r to the lethal effects of UV light (by a factor of 33), γ-radiation (55), NG (15) and NA (62), showed intermediate resistance to MTC (4) and HA(7), but was sensitive to EMS (1) and βPL (2). M. radiodurans was very resistant to mutagens producing damage which can be repaired by a recombination system, indicating that it possesses an extremely efficient recombination repair mechanism.Both species were equally sensitive to mutation to trimethoprim resistance by NG, but M. radiodurans was more resistant the E. coli B/r to the other multagens tested, being non-mutable by UV light, γ-radiation, MTC and HA, and only slightly sensitive to mutation by NA, EMS, and βPL. The resistance of M. radiodurans to mutation by UV-light, γ-radiation and MTC is consistent with an hypothesis that recombination repair in M. radiodurans is accurate since these mutagens may depend on an “error-prone” recombination system for their mutagenic effect in E. coli B/r. However, because M. radiodurans is also resistant to mutagens such as HA and EMS, which are mutagenic in E. coli in the absence of an “error-prone” system, we propose that all the mutagens tested may have a common mode of action in E. coli B/r, but that this mutagenic pathway is missing in M. radiodurans.     

Effects of DNA damaging agents on cultured fibroblasts derived from patients with Cockayne syndrome.

The cytotoxic action of physical and chemical agents on 10 skin fibroblast strains in culture derived from individuals with Cockayne's syndrome was measured in terms of colony-forming ability. As compared to fibroblasts from normal donors, all Cockayne cell strains tested exhibited a significantly increased sensitivity to UV light and a normal sensitivity to X-rays. Cells from two sets of parents of unrelated Cockayne children showed an intermediate level of UV sensitivity. There was no effect of 0.5 mM caffeine on UV survival in normal and two Cockayne strains tested, indicating that postreplicational repair in Cockayne cells as measured by caffeine sensitivity was probably normal. Sensitivity of normal and Cockayne cells to the chemical carcinogens and mutagens 4NQO, N-AcO-AAF, ICR-170 and EMS was also compared. An increased sensitivity of Cockayne cells to 4NQO or N-AcO-AAF, but not the ICR-170 or EMS, was observed. However, unlike the intermediate UV sensitivity, the cell strains from two parents of Cockayne patients showed the same sensitivity to N-AcO-AAF or 4NQO as fibroblasts from normal individuals. Quantiation of damage to the DNA after 20 J . m-2 UV irradiation indicates normal levels of [3H] thymidine incorporation in the Cockayne cells, in contrast to UV-irradiated xeroderma pigmentosum cells (XP 12BE) in which there was a very low level of repari synthesis. Moreover, we have shown previously that excision of UV-induced pyrimidine dimers in 2 of the 10 Cockayne cell strains was normal.     

Antimutagenic effect of garlic (Allium sativum L.) on 4NQO-induced mutagenesis in Escherichia coli WP2

Aqueous extract prepared from garlic bulbs markedly suppressed the mutagenesis in both E. coli WP2 trp- and E. coli WP2 trp- uvrA- induced by 4-nitroquinoline 1-oxide (4NQO), but not that induced by UV. Cellular toxicity, inhibition of the expression of the Trp+ phenotype and delay of the first cell division after 4NQO treatment were not observed in the presence of the extract. Since the extract showed identical antimutagenic effects against 4NQO in both test strains but no effect on the mutagenesis of UV, it seems that the extract might act by inactivating the electrophilic group(s) of 4NQO or inhibiting its metabolic activation.     

Mutagenic and error-free DNA repair in Streptomyces

Summary Two mutants of Streptomyces fradiae defective in DNA repair have been characterized for their responses to the mutagenic and lethal effects of several chemical mutagens and ultraviolet (UV) light. S. fradiae JS2 (mcr-2) was more sensitive than wild type to agents which produce bulky lesions resulting in large distortions of the double helix [i.e. UV-light, 4-nitroquinoline-1-oxide (NQO), and mitomycin C (MC)] but not to agents which produce small lesions [i.e. hydroxylamine (HA), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and N-methyl-N-nitro-N-nitrosoguanidine (MNNG)]. JS2 expressed a much higher frequency of mutagenesis induced by UV-light at low doses and thus appeared to be defective in an error-free excision repair pathway for bulky lesions analogous to the uvr ABC pathway of Escherichia coli. S. fradiae JS4 (mcr-4) was defective in repair of damage by most agents which produce small or bulky lesions (i.e., HA, NQO, MMS, MNNG, MC, and UV, but not EMS). JS4 was slightly hypermutable by EMS and MMS but showed reduced mutagenesis by NQO and HA. This unusual phenotype suggests that the mcr-4 ⁺ protein plays some role in error-prone repair in S. fradiae.     

Bio-antimutagenic effects of tannic acid on UV and chemically induced mutagenesis in Escherichia coli B/r

Tannic acid suppressed the mutagenesis in E. coli B/r WP2 trp- induced by UV or 4-nitroquinoline 1-oxide (4NQO), but not that induced by gamma-rays or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The depression of mutations induced by UV was most remarkable in the DNA-repair-proficient strain (WP2). Tannic acid, however, showed no bio-antimutagenic effect in the excision repair-deficient strain (WP2s uvrA- or ZA159 uvrB-) under the test conditions where no cellular toxicity was observed. The effect ceased within 30 min after UV irradiation. The inhibition of the expression of Trp+ phenotype and the delay of the first cell division after UV irradiation were not observed in the presence of tannic acid. From these results we conclude that tannic acid may enhance the excision-repair system probably by activating the repair enzymes or by interacting with DNA.     

Mutagen detection with a mouse line containing 3 distinct mutations conferring sensitivity

A mouse-cell mutant sensitive to methyl methanesulfonate (MMS), X-rays, ultraviolet light (UV), and crosslinking agents was selected using the replica plating and cell suspension spotting methods. This mutant (XUM1) is a mitomycin C-sensitive derivative of previously reported XU1, a mutant sensitive to MMS, X-rays and UV. Since XU1 is highly susceptible to the lethal effect of 4-nitroquinoline-1-oxide (4NQO), XUM1 is also hypersensitive to 4NQO. Growth inhibition area tests showed that low concentrations of mutagens were detected with the multiple mutagen-sensitive mutant XUM1. Hence XUM1 cells will be useful in detecting with high sensitivity a wide range of mutagens and carcinogens which mimic X-rays, UV and crosslinking agents.     

The superiority of hamster liver microsomal fraction for activating nitrosamines to mutagens in Salmonella typhimurium

46 chemicals of various classes and structures, including 30 known animal carcinogens, were evaluated for genotoxic effects using the Escherichia coli rec assay with strains WP2 (wild-type) and WP100 (uvrA- recA-) in qualitative and quantitative spot tests and in quantitative suspension tests. The rec assay detected 17 of 30 known carcinogens as genotoxic agents, including mitomycin C and diethylnitrosamine, both negative in the Salmonella/Ames test as utilized in these studies. The rec assay in conjunction with the Salmonella/Ames test detected 20 of 30 known carcinogens as genotoxic agents. Azo/aminoazo carcinogens showed little gentoxicity, and the aromatic amine 2-acetylaminofluorene was non-genotoxic in the rec assay. The rec assay was more effective than pol tests with E. coli strains W3110/p3478 and strains WP2/WP67. Effectiveness of the rec assay was related to the DNA repair-defective nature of the uvrA- recA- genotype of strain WP100.     

The roles of different excision-repair mechanisms in the resistance of Micrococcus luteus to UV and chemical mutagens

M. luteus mutants showing increased sensitivity to both UV and 4-NQO were isolated after the treatment of parental ATCC4698 strain with MNNG. The mutants were also highly sensitive to mitomycin C, cis-platinum, 8-methoxypsoralen (8-MOP) plus near-UV and angelicin plus near-UV in various degrees. The endonuclease activity specific for pyrimidine dimers in UV-irradiated DNA was normally detected in extract of the mutants. With regard to host-cell reactivation ability the mutants fell into two groups. The hcr- mutants lacked the ability to reactivate UV-damaged N6 phage and were resistant to X-rays. The incision of DNA did not occur during incubation after the treatment with angelicin plus near-UV in the hcr- mutants, whereas it occurred in the parental strain. The facts indicate that the hcr- mutants are defective in the incision mechanism which has a wide substrate specificity, similar to the UVRABC nuclease of E. coli. On the other hand, the incision of DNA and the removal of UV-induced thymine dimers from DNA occurred in the hcr- mutants as well as in the parental strain, which is ascribed to the UV endonuclease activity. Compared with the hcr- mutants, hcr+ mutants were highly sensitive to X-rays, like recA- mutants of E. coli.     

Changes in the survival curve shape of E. coli cells following irradiation in the presence of uncouplers of oxidative phosphorylation

Four uncouplers of oxidative phosphorylation (UOP) (carbonyl cyanide m-chlorophenylhydrazone, 2,4-dinitrophenol, 4-hydroxybenzylidenemalonitrile and N-phenylanthranilic acid) have been found to alter the shape of the radiation survival curves of several cell lines of E. coli when present during irradiation in oxia. Incubation of cells with high concentrations of UOP for 30 min before irradiation induced an increase in extrapolation number (n) in cell lines AB 1157 (wild-type), AB 1886(uvrA-) and KMBL(polA-) but not GR 501(lig-)ts, AB 2463(recA-) and AB 2480(uvrA-recA-). In addition the UOP all effect a decrease in mean lethal dose (D0) even when tested at low concentrations or short contact times. Studies with wild-type cells correlate the increase in n with measured increased levels of ATP (above oxic control cells) produced upon incubation with UOP. The increased levels of ATP most likely arise from the UOP overstimulating glycolysis. The decrease in D0 cannot be associated with any of the repair pathways investigated and it is concluded that the highly lipophilic UOP directly or indirectly potentiate other target(s) to radiation damage as well as DNA under oxic conditions. Treatment of the cells with UOP did not result in the deleterious depletion of energy substrates, loss of non-protein thiols or the production of cytotoxins upon irradiation.     

Genetic Evidence that the uvsE Gene Product of Deinococcus radiodurans R1 Is a UV Damage Endonuclease

An in vitro transposition system, developed to facilitate gene disruption in Deinococcus radiodurans R1, has been used to inactivate the gene designated dr1819 in uvrA-1(+) and uvrA-1 backgrounds. dr1819 encodes a protein with homology to a UV DNA damage endonuclease expressed by Schizosaccharomyces pombe. Interruption of dr1819 greatly sensitizes the uvrA-1 strain but not the uvrA-1(+) strain to UV light, indicating that the dr1819 gene product is a component in a DNA repair pathway that can compensate for the loss of nucleotide excision repair in this species. Clones of dr1819 will restore UV resistance to UVS78, a uvrA-1 uvsE strain, indicating that dr1819 and uvsE are the same locus.     

Effect of DNA Repair systems on antibacterial and mutagenic activity of an antitumor protein, neocarzinostatin.

Antibacterial and mutagenic effects of an antitumor protein, neocarzinostatin (NCS), on isogenic strains of Escherichia coli with normal or defective DNA repair systems were studied. Growth of the strains lacking recA gene was inhibited by NCS with much lower concentration than in the case of those possessing it, while the "differential inhibition for growth" (DIG) between the strains uvrA+ and uvrA- was not seen. NCS induces mutation in recA+ strains but not significantly in recA-, while no such difference of mutagenesis was noticed between the strains uvrA+ and uvrA-. These results suggest that NCS produces hardly excisable DNA damage which is repaired by an error-prone recombination process.