The formation of photoreactivable damage by direct excitation of DNA in X-irradiated E. coli cells

The role of the direct excitation process in the formation of photoreactivable damage (pyrimidine dimers) in E. coli WP2 hcr-exr- cells has been studied. The pyrimidine dimers were detected by photoreactivation following anoxic irradiation by X-rays (220 kVp). The dose modifying factor (DMF) is 1.28 +/- 0.09. A biophysical model is used for a theoretical examination of the importance of the direct excitation process in the formation of photoreactivable damage and the experimental data are consistent with this model.     

Spectrum of formation of photo- and non-photoreactivated damage in E. coli cells irradiated with UV light (250-334 nm)

A relative contribution of photoreactivated (modified by visible light) and non-photoreactivated (modified by temperature) damages to UV-irradiated (250-334 nm) E. coli B cells was estimated. The contribution of damages modified by temperature to a lethal effect of UV-radiation was invariable within the range from 250 to 334 nm. The photoreactivation of E. coli B cells was also independent of lambda-inactivating UV-light within 250-313 nm, and its value exceeded that of the wild-type E. coli WP2 which did not vary by the mode of UV-damages repair. Moreover, in contrast to E. coli B. cells, the value of the photoreactivation of E. coli WP2 decreased, as lambda-inactivating UV-light increased from 250 to 313 nm.     

Cross-adaptive response in Escherichia coli caused by pretreatment with H2O2 against formaldehyde and other aldehyde compounds.

A cross-adaptive response (CAR), defined as a reduction of the effects of an agent by pretreatment with another agent, was demonstrated when E. coli WP2 cells were pretreated with hydrogen peroxide (H2O2) followed by challenging treatment with aldehyde compounds. Pretreatment with a sublethal dose (60 microM) of H2O2 for 30 min made WP2 cells resistant to the killing effects of formaldehyde (FA), and 4 other mutagenic aldehydes: glutaraldehyde, glyoxal, methyl glyoxal and chloroacetaldehyde. CAR was also observed in WP2uvrA (uvrA-) and ZA12 (umuC-) cells, but not in ZA60 (recA-) and CM561 (lexA- (Ind-] cells. A role of recA and lexA in CAR was further suggested by the lack of beta-galactosidase induction in recA- and lexA- cells by H2O2. CAR and beta-galactosidase induction, however, were found to be separate events since CAR was recovered by introducing the recA+ gene into lexA- cells, but no induction of beta-galactosidase by H2O2 was observed in cells with the same gene transfer. These results suggest that H2O2 has the capacity to induce a function which reduces the killing effects of aldehydes, and the function is controlled by the recA gene without involvement of SOS response.     

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.     

Effect of mutations in the uvrA and recA genes on the photoreactivity of Escherichia coli cells irradiated by UV light (250 and 313 nm)

The relative contribution of photo- and non-photoreactivable damages to the lethal effect of far-(250 nm) and mid-(313 nm) wave UV in isogenic bacterial cells Escherichia coli WP2 (wild type, uvrA and recA mutants) was estimated. It has been demonstrated that the value of non-photoreactivable damages increases with lambda of UV (250----313 nm) and depends on the genotype (uvrA and recA).     

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).     

Genetic determination and mechanism of realization of temperature-induced radioresistance

A study was made of the influence of the repair genotype of E. coli cells on the realization of the effect of enhanced radioresistance during gamma-irradiation at elevated temperatures (40-45 degrees C). The effect of the thermoinduced radioresistance (TIR) was diminished significantly but not abolished completely in mutant cells selectively deficient in excision or recombination repair systems (po1A1, recB21C22sbcB15, recF143 mutants). However mutations which exclude the recA gene product (recA13, recA13B21C22 or lexA3 mutants) inhibited TIR completely. The introduction of recA+ gene into recA- or lexA- mutants almost normalized TIR. On the basis of the data obtained the authors discuss the role of recA protein in activation of the membrane-associated repair complex whose efficiency depends on the temperature of gamma-irradiation.     

Radioadaptive enhancement of the repair of UV-induced postreplication gaps in Escherichia coli cells deficient in DNA repair

In UV-irradiated E. coli WP2 uvrA, deficient in excision repair of DNA with pyrimidine dimers, gamma-irradiation in low doses (radioadaptation) before UV-irradiation leads to the intensification of postreplication repair of DNA. This process in WP2 uvrA polA and uvrA lexA mutants is less than in WP2 uvrA cells, but in WP2 uvrA recA both postreplication repair and its radioadaptive intensification are absent. In E. coli AB1157 excising pyrimidine dimers the radioadaptive intensification of postreplication repair of DNA is expressed almost to the same extent as in WP2 uvrA. In GW2100 umuC mutant, deficient in DNA polymerase V, postreplication repair of DNA is expressed, but its radioadaptive intensification is absent, while in AB2463 recA13 both postreplication repair of DNA and radioadaptive intensification of postreplication repair of DNA are absent. The above data suggest that DNA polymerase I and LexA protein are needed for radioadaptive intensification of postreplication repair of DNA in uvrA strain, and DNA polymerase V is needed for radioadaptive intensification in E. coli AB1157, and that RecA protein is required for postreplication repair and radioadaptive intensification of postreplication repair of DNA.     

Influence of the recA- mutation on spontaneous filamentation and the titer of the lambda phage in ylonB- mutants of E. coli K12 (lambda)]

A study was made of the effect of recA- mutation on the spontaneous filament formation and the spontaneous induction of the lambda prophage in the E. coli K12 strain bearing lonB- mutation. It was revealed that in the absence of a functionally-active product of the recA- gene the lonB- strain of E. coli K12 displayed but a partial depression of spontaneous formation of filamentous forms and of spontaneous induction of the lambda prophage.     

Induction of radioresistance in Escherichia coli.

The effect of prior treatment by inducing agents on the radioresistance of cells of Escherichia coli has been studied. In order to separate the induction process from the radiation-damage process, cells were first treated with inducing agents such as ultraviolet light, ionizing radiation, or nalidixic acid, allowed to become induced by incubation for 50 min and then given rifampin to prevent further induction. They were then tested for radiation sensitivity. It was found that all strains tested except recA-, lex-, and recB showed very apparent protection. Induction by UV had the most effect and by nalidixic acid the least. The time course of development of protection was observed in one case: it is 50% established in 15 min. The absence of effect in recA- and lex- is explainable by the fact that these cells cannot be induced, for example, for prophage or the inducible inhibitor of post-irradiation DNA degradation. We suggest that the inducible inhibitor of postirradiation DNA degradation is one factor in a recovery system possessed by E. coli cells.     

Mechanistic analysis of the contributions of DNA and protein damage to radiation-induced cell death

Protein oxidation can contribute to radiation-induced cell death by two mechanisms: (1) by reducing the fidelity of DNA repair, and (2) by decreasing cell viability directly. Previously, we explored the first mechanism by developing a mathematical model and applying it to data on Deinococcus radiodurans . Here we extend the model to both mechanisms, and analyze a recently published data set of protein carbonylation and cell survival in D. radiodurans and Escherichia coli exposed to gamma and ultraviolet radiation. Our results suggest that similar cell survival curves can be produced by very different mechanisms. For example, wild-type E. coli and DNA double-strand break (DSB) repair-deficient recA- D. radiodurans succumb to radiation doses of similar magnitude, but for different reasons: wild-type E. coli proteins are easily oxidized, causing cell death even at low levels of DNA damage, whereas proteins in recA- D. radiodurans are well protected from oxidation, but DSBs are not repaired correctly even when most proteins are intact. Radioresistant E. coli mutants survive higher radiation doses than the wild-type because of superior protection of cellular proteins from radiogenic oxidation. In contrast, wild-type D. radiodurans is much more radioresistant than the recA- mutant because of superior DSB repair, whereas protein protection in both strains is similar. With further development, the modeling approach presented here can also quantify the causes of radiation-induced cell death in other organisms. Enhanced understanding of these causes can stimulate research on novel radioprotection strategies.     

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.     

A comprehensive exercise in comparative mutagenesis with exciting outcome or how good are mutation assays in predicting carcinogens?

Cultivation of E. coli B/r strain WP2 in low concentrations of either 4-nitroquinoline N-oxide (4NQO) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) had no effect on the mutagenic or cytotoxic consequences of subsequent challenge with dichlorvos (DCV). However, although the sensitivity of E. coli cells taken from cultures grown in low concentrations of DCV to the effects of 4NQO was unchanged, the cells were more resistant to the mutagenic (but not cytotoxic) consequences of MNNG challenge. This phenomenon was not observed in WP2 derivatives deficient in either error-free (uvrA-) or error-prone (lexA-) DNA-repair, suggesting that a factor common to both these repair pathways may be involved.     

Formation of plasmid pBR322 oligomers as depending on a tetracycline concentration

Escherichia coli K12 strains containing the plasmid pBR322 often show varying contents of plasmid oligomers, in which the monomer units are arranged in tandem. When the concentration of the plasmid-selective antibiotic tetracycline in the medium becomes increased selection of cells containing largely higher oligomers occurs. The number of monomer units organized in the oligomers increases with tetracycline concentration. recA- mutants are unable to generate oligomers under the same conditions and show lower tetracycline resistance. This observations suggest a selective advantage of oligomer containing cells in the presence of tetracycline as a result of higher gene dosage. But E. coli cells transformed with monomers, dimers, trimers, as well as tetramers of pBR322 are characterized by roughly the same plasmid DNA content as well as plasmid coded beta-lactamase and resistance to tetracycline.     

Repair of UV-irradiated plasmid DNA in mutants of Saccharomyces cerevisiae and Escherichia coli deficient in repair of pyrimidine dimers

The repair of in vitro UV-irradiated DNA of plasmid pBB29 was studied in excision defective yeast mutants rad1, rad2, rad3, rad4, rad10 and in Escherichia coli mutants uvr- and recA-, by measuring the cell transformation frequency. Rad2, rad3, rad4, and rad10 mutants could repair plasmid DNA despite their inability to repair nuclear DNA, whereas the reduced ability of rad1 mutant for plasmid DNA repair demonstrated alone the same dependence on the host functions that are needed for nuclear DNA repair. In E. coli the repair of UV-irradiated plasmid DNA is carried out only by the excision-repair system dependent on uvr genes. Treatment of UV-irradiated plasmid DNA with UV endonuclease from Micrococcus luteus greatly enhances the efficiency of transformation of E. coli uvr- mutants. Similar treatment with cell-free extracts of yeast rad1 mutant or wild-type strains as well as with nuclease BaL31, despite their ability for preferential cutting of UV damaged DNA, showed no influence on cell transformation.     

Contribution of a photoreactivable component to the oxygen effect observed in certain rad mutants of Saccharomyces cerevisiae after exposure to high energy electrons

It is shown that a fraction of damage induced by high energy electrons (25 MeV) in certain rad mutants of the yeast Saccharomyces cerevisiae can be photoreactivated. The photoreactivable damage contributes to the lethal effect of this type of irradiation and modifies the oxygen effect. Using photoreactivating light or nigrosin, the amount of photoreactivable damage is reduced and the oxygen enhancement ratio (OER) for yeast mutants increases approximately to the OER found in wild-type cells.     

Microbioluminescent study of the general toxicity and mutagenicity of pollutants

Bacterial bioluminescence was applied to detection of general toxicity (MIT test) and genotoxicity (SOS-lux test) of some chemicals, seawater, and fresh water. The SOS-induced luminescence of E. coli WP2s (cda::luxCDABE) cells was higher than in E. coli C 600 (cda::luxCDABE) at 37 degrees C and pH 6.5. The mutagenic effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), mitomycin C, and hydrogen peroxide determined from the induction of E. coli WP2s cell luminescence was detected at lower concentrations than in the assessment of reversion frequencies. General toxicity was demonstrated by using luminescence inhibition for hydrogen peroxide, Zn2+, and Cd2+ at low concentrations. Regions of the Krasnodar Krai where sea and fresh waters exerted toxic action on luminescence were determined by the microbioluminescent method.     

Starvation as an inducer of error-free DNA repair in Escherichia coli

Previous work in this laboratory has shown that heat shock or vitamin B1 deprivation induces an error-free DNA-repair process in Escherichia coli. The system is absolutely dependent on excision repair, while its induction is delayed in lon- or recA- cells. We have now shown that starvation of E. coli for amino acids, glucose or phosphate, conditions known to induce the stringent response or the glu and pho regulons, respectively, leads to a similar uvrA-dependent increase in UV resistance and decrease in UV-induced mutation frequency. These results support the hypothesis that the effect is a general response to non-mutagenic stress that may play an important role in the survival of cells exposed to harsh environments.     

Analysis of the antimutagenic effect of cinnamaldehyde on chemically induced mutagenesis in Escherichia coli

The antimutagenic effect of cinnamaldehyde on mutagenesis was investigated using ten kinds of chemical mutagen in Escherichia coli WP2s (uvr A-). In addition, the frequency of mutation induction by each mutagen in an SOS repair deficient (umuC-) strain was compared with that in a wild-type (umuC+) strain. Cinnamaldehyde greatly suppressed the umuC-dependent mutagenesis induced by 4-nitroquinoline 1-oxide (4-NQO), furylfuramide or captan. However, cinnamaldehyde was less effective against the umuC-independent mutagenesis by alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine and ethylmethanesulfonate. On the other hand, no inhibitory effect of cinnamaldehyde was observed on prophage induction or tif-mediated filamentous growth. These results suggest that a cinnamaldehyde does not prevent the induction of the SOS functions. Despite the decrease in the number of revertants, a remarkable increase was observed in the survival of 4-NQO-treated WP2s cells after exposure to cinnamaldehyde. The reactivation of survival suggests the promotion of some DNA repair system by cinnamaldehyde. This enhancement of survival was also observed in uvr B, polA, recF or umuC mutants and less in lexA or recB, C mutants. However, it was not observed in recA mutants. Therefore, we assume that cinnamaldehyde may enhance an error-free recombinational repair system by acting on recA-enzyme activity.     

A 50 Hz, 14 mT magnetic field is not mutagenic or co-mutagenic in bacterial mutation assays

We used bacterial mutation assays to assess the mutagenic and co-mutagenic effects of power frequency magnetic fields (MF). For the former, we exposed four strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537) and two strains of Escherichia coli (WP2 uvrA, WP2 uvrA/pKM101) to 50Hz, 14mT circularly polarized MF for 48h. All results were negative. For the latter, we treated S. typhimurium (TA98, TA100) and E. coli (WP2 uvrA, WP2 uvrA/pKM101) cells with eight model mutagens (N-ethyl-N'-nitro-N-nitrosoguanidine, 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide, 4-nitroquinoline-N-oxide, 2-aminoanthracene, N(4)-aminocytidine, t-butyl hydroperoxide, cumen hydroperoxide, and acridine orange) with and without the MF. The MF induced no significant, reproducible enhancement of mutagenicity. We also investigated the effect of MF on mutagenicity and co-mutagenicity of fluorescent light (ca. 900lx for 30min) with and without acridine orange on the most sensitive tester strain, E. coli WP2 uvrA/pKM101. Again, we observed no significant difference between the mutation rates induced with and without MF. Thus, a 50Hz, 14mT circularly polarized MF had no detectable mutagenic or co-mutagenic potential in bacterial tester strains under our experimental conditions. Nevertheless, some evidence supporting a mutagenic effect for power frequency MFs does exist; we discuss the potential mechanisms of such an effect in light of the present study and studies done by others.