Toxicity and SOS-response to ELF magnetic fields and nalidixic acid in E. coli cells

Extremely low-frequency magnetic fields (ELF-MF) have previously been shown to affect conformation of chromatin and cell proliferation. Possible genotoxic and carcinogenic effects of ELF-MF have also been discussed and tested. In this study, we analysed the effect of ELF-MF on chromatin conformation in E. coli GE499 cells by the anomalous viscosity time-dependence (AVTD) technique. Possible genotoxic effects of the specific combination of static and ELF-MF, which has been proven to affect chromatin conformation, were investigated by a clonogenic assay, by assessing cell-growth kinetics, and by analysis of the SOS-response by means of inducible recA-lacZ fusion-gene products and the β-galactosidase assay. The genotoxic agent nalidixic acid (NAL) was used as a positive control and in combination with ELF-MF. Nalidixic acid at 3-30μg/ml decreased the AVTD peaks and induced a cytotoxic effect. In contrast to NAL, ELF-MF fields increased AVTD, stimulated cell growth, and increased cloning efficiency. These effects depended on the frequency within the range of 7-11Hz. While NAL induced an SOS-response, exposure to ELF-MF did not induce the recA-lacZ fusion-gene product. Exposure to ELF-MF did not modify the genotoxic effects of NAL either. All together, the data show that ELF-MF, under specific conditions of exposure, acted as a non-toxic but cell-growth stimulating agent.     

Frequency-dependent effects of ELF magnetic field on chromatin conformation in Escherichia coli cells and human lymphocytes.

The effects of magnetic fields of extremely low frequency (ELF, 21 microT r.m.s.) on cells of different Escherichia coli K12 strains and human lymphocytes were studied by the method of anomalous viscosity time dependence (AVTD). Within the frequency range of 6-24 Hz, two resonance-type frequency windows with maximal effects at 9 Hz and 16 Hz were observed in response of GE499 strain. Only one frequency window with maximum effect at 8.5 Hz was found for GE500 cells. These data along with previously obtained for two other E. coli strains, AB1157 and EMG2, indicate that frequency windows are dependent on genotype of cells exposed to ELF. Resonance-type effects of ELF were also observed in human lymphocytes in frequency windows around 8 and 58 Hz. These ELF effects differed significantly between studied donors, but were well reproducible in independent experiments with lymphocytes from the same donors. The frequency windows in response of E. coli strains and human lymphocytes to ELF significantly overlapped suggesting that the same targets may be involved in this response. We compared the frequency windows with predictions based on the ion cyclotron resonance (ICR) model and the magnetic parametric resonance model. These models predicted effects of ELF magnetic fields at the 'cyclotron' frequencies of some ions of biological relevance. According to the ICR model, ELF effects should be also observed at harmonics of cyclotron frequencies and, contrary, parametric resonance model predicted effects at subharmonics. While we observed coincidence of each experimental resonance frequency with predictions of one of these two models, all experimentally defined effective frequency windows were in good agreement with relatively narrow frequency ranges of both harmonics and subharmonics for natural isotopes of Na, K, Ca, Mg, and Zn ions. The experimental data support idea that both harmonics and subharmonics of several biologically important ions are involved in frequency-dependent ELF effects in cells of different types.     

R-plasmid transfer and its response to nalidixic acid.

The conjugational transfer efficiency of 41 wild-type R-plasmids was studied in Escherichia coli K-12. Type I R-plasmids were transferred at comparatively high and rather uniform frequencies, whereas type F R-plasmids showed less uniform and, on average, somewhat lower transfer frequencies. R-plasmids not mediating sensitivity to F-, I-, or N-specific phages showed moderate transfer frequencies, and type N R-plasmids showed very low transfer frequencies. Various lines of evidence suggest that a well-expressed, but functionally inefficient, conjugation apparatus is the cause of the poor transfer of type N R-plasmids in liquid medium. Nalidixic acid efficiently inhibited transfer of type I and particularly type F R-plasmids, whereas the transfer of type N plasmids was resistant to the drug. Type F and type I plasmids appear to depend on at least one host function for their transfer, namely, the nalidixic acid-sensitive reaction in vegetative chromosome replication, whereas type N plasmids are independent of this function.     

Regulatory role of recF in the SOS response of Escherichia coli: impaired induction of SOS genes by UV irradiation and nalidixic acid in a recF mutant

We isolated a new recF mutant of Escherichia coli K-12 by insertion of transposon Tn5 into the recF gene. This recF400::Tn5 allele displayed the same phenotypic characteristics as the classic recF143 mutation. By using Mu d(Ap lac) fusions, the induction of nine SOS genes, including recA, umuC, dinA, dinB, dinD, dinF, recN, and sulA, by UV irradiation and nalidixic acid was examined. Induction of eight genes by the two agents was impaired by recF400::Tn5 to different extents. The ninth fused SOS gene, dinF, was no longer inducible by UV when combined with recF400::Tn5. The generally impaired SOS response in recF strains did not result from weak induction of recA protein synthesis, since a recA operator-constitutive mutation did not alleviate the inhibitory effect of the recF mutation. The results suggest that recF plays a regulatory role in the SOS response. It is proposed that this role is to optimize the signal usage by recA protein to become a protease.     

Suppression of the E. coli SOS response by dNTP pool changes

The Escherichia coli SOS system is a well-established model for the cellular response to DNA damage. Control of SOS depends largely on the RecA protein. When RecA is activated by single-stranded DNA in the presence of a nucleotide triphosphate cofactor, it mediates cleavage of the LexA repressor, leading to expression of the 30⁺-member SOS regulon. RecA activation generally requires the introduction of DNA damage. However, certain recA mutants, like recA730, bypass this requirement and display constitutive SOS expression as well as a spontaneous (SOS) mutator effect. Presently, we investigated the possible interaction between SOS and the cellular deoxynucleoside triphosphate (dNTP) pools. We found that dNTP pool changes caused by deficiencies in the ndk or dcd genes, encoding nucleoside diphosphate kinase and dCTP deaminase, respectively, had a strongly suppressive effect on constitutive SOS expression in recA730 strains. The suppression of the recA730 mutator effect was alleviated in a lexA-deficient background. Overall, the findings suggest a model in which the dNTP alterations in the ndk and dcd strains interfere with the activation of RecA, thereby preventing LexA cleavage and SOS induction.     

The nature of SOS response in E. coli K-12 (uvrA) cells exposed to the different ultraviolet doses

The kinetic and dose dependencies of the SOS-induction in E. coli (uvrA) cells exposed to UV light were investigated. Below 2 J/m2 the rate of the SOS-induction increased with dose. The maximal level of the SOS-response was proportional to the UV dose. Pyrimidine dimers were necessary for the induction. In the dose range 2-10 J/m2 the rate of the SOS-induction decreased with dose. The dose-response curve was non-linear. Pyrimidine dimers were not required for the induction. The nature of the molecular events leading to the SOS-induction at low and high doses was discussed.     

Penetration of nalidixic acid into Escherichia coli K-12 cells

Transport of nalidixic acid (NAL) into Escherichia coli cells subjected to osmotic shock, permeabilised with toluene or treated with DNP, CCCP or EDTA, was studied. It was found that osmotic shock and protonophores do not inhibit the transport of [3H]NAL, however, the transport of [3H]DAP and [3H]glucose is reduced. EDTA and toluene enhance penetration of [3H]NAL. This effect is, however, abolished in the presence of Mg++ ions. It is suggested that NAL penetrates into the cell by simple or facilitated diffusion and that the outer membrane of E. coli is the penetration barrier for the drug.     

Fluctuation analysis of mutations to nalidixic acid resistance in Escherichia coli.

Mutations of Escherichia coli from sensitivity to nalidixic acid resistance were studied by fluctuation analysis. The mutant distributions in replicate cultures were not significantly affected either by the age of the carbon-starved preculture used for inocula or by the inoculum size. The data from 23 fluctuation tests (48 cultures each) were pooled. The mean number of mutations per culture was estimated to be 0.71 from the fraction of cultures without mutants or 0.74 and 0.77 by maximum-likelihood estimation based on the two models under consideration. When the pooled data were compared with the theoretical expectations, the fits were unsatisfactory (P < 0.005). The lack of fit was caused mainly by too high a frequency of cultures with between 17 and 32 mutants and too high a frequency of cultures with more than 128 mutants. Possible reasons for the lack of fit and its implications with respect to estimation of mutation rates from fluctuation tests are discussed.     

The epsilon subunit of DNA polymerase III Is involved in the nalidixic acid-induced SOS response in Escherichia coli

Quinolone antibacterial drugs such as nalidixic acid target DNA gyrase in Escherichia coli. These inhibitors bind to and stabilize a normally transient covalent protein-DNA intermediate in the gyrase reaction cycle, referred to as the cleavage complex. Stabilization of the cleavage complex is necessary but not sufficient for cell killing--cytotoxicity apparently results from the conversion of cleavage complexes into overt DNA breaks by an as-yet-unknown mechanism(s). Quinolone treatment induces the bacterial SOS response in a RecBC-dependent manner, arguing that cleavage complexes are somehow converted into double-stranded breaks. However, the only proteins known to be required for SOS induction by nalidixic acid are RecA and RecBC. In hopes of identifying additional proteins involved in the cytotoxic response to nalidixic acid, we screened for E. coli mutants specifically deficient in SOS induction upon nalidixic acid treatment by using a dinD::lacZ reporter construct. From a collection of SOS partially constitutive mutants with disruptions of 47 different genes, we found that dnaQ insertion mutants are specifically deficient in the SOS response to nalidixic acid. dnaQ encodes DNA polymerase III epsilon subunit, the proofreading subunit of the replicative polymerase. The deficient response to nalidixic acid was rescued by the presence of the wild-type dnaQ gene, confirming involvement of the epsilon subunit. To further characterize the SOS deficiency of dnaQ mutants, we analyzed the expression of several additional SOS genes in response to nalidixic acid using real-time PCR. A subset of SOS genes lost their response to nalidixic acid in the dnaQ mutant strain, while two tested SOS genes (recA and recN) continued to exhibit induction. These results argue that the replication complex plays a role in modulating the SOS response to nalidixic acid and that the response is more complex than a simple on/off switch.     

Interspecies regulation of the SOS response by the E. coli lexA+ gene

A plasmid-encoded E. coli lexA+ gene was introduced into 6 species of Enterobacteria. Ultraviolet light-sensitization occurred in all species except P. rettgeri, and 4 organisms showed reduced inducibility of RecA-like proteins. The mechanism of lexA+ control of the SOS response therefore appears common to several species.     

Effect of static magnetic field on E. coli cells and individual rotations of ion-protein complexes

The effect of week static magnetic fields on Escherichia coli K12 AB1157 cells was studied by the method of anomalous viscosity time dependencies (AVTD). The AVTD changes were found when E. coli cells were exposed to static fields within the range from 0 to 110 microT. The dependence of the effect on the magnetic flux density had several extrema. These results were compared with theoretical predictions of the ion interference mechanism. This mechanism links the dissociation probability of ion--protein complexes to parameters of magnetic fields. The mechanism was extended to the case of rotating complexes. Calculations were made for several ions of biological relevance. The results of simulations for Ca(2+), Mg(2+), and Zn(2+) showed a remarkable consistency with experimental data. An important condition for this consistency was that all complexes rotate with the same speed approximately 18 revolutions per second (rps). This suggests that the rotation of the same carrier for all ion--protein complexes may be involved in the mechanism of response to the magnetic field. We believe that this carrier is DNA.     

Mutations in uvrD induce the SOS response in Escherichia coli.

We have isolated three new mutations in uvrD that increase expression of the Escherichia coli SOS response in the absence of DNA damage. Like other uvrD (DNA helicase II) mutants, these strains are sensitive to UV irradiation and have high spontaneous mutation frequencies. Complementation studies with uvrD+ showed that UV sensitivity and spontaneous mutator activity were recessive in these new mutants. The SOS-induction phenotype, however, was not completely complemented, which indicated that the mutant proteins were functioning in some capacity. The viability of one of the mutants in combination with rep-5 suggests that the protein is functional in DNA replication. We suggest that these mutant proteins are deficient in DNA repair activities (since UV sensitivity is complemented) but are able to participate in DNA replication. We believe that defective DNA replication in these mutants increases SOS expression.     

Physiological changes in adult rats exposed to an ELF rotating magnetic field

In three Experiments (I, II, IV), adult male rats, between 115 and 150 days of age were exposed to either a 0.5–3 or 3–30 gauss ELF (0.5 Hz) rotating magnetic field (RMF), for 5, 10, or 26 days. The rats exposed to the RMF for 10 and 26 days averaged significantly (p< 0,05) greater water consumption than controls. The group exposed to the RMF for 5 days also consumed more water, although the difference was not significant. ELF-RMF-exposed rats also showed a progressive decrease (p <0.02) in relative thyroid weights, but increase in body weight gain (p <0.001) up to 10 days of exposure, and increase in testicle weights (p < 0.05) up to 26 days of exposure. No significant differences were found between groups for circulating blood eosinophil counts or relative adrenal weights, although again the differences did increase with duration of exposure. In a fourth Experiment (III), rats that were 80 days of age at the beginning of 21 days of exposure did not show any significant differences in the above measures from the control group. The changes in behavior and physiology associated with ELF exposure is discussed in terms of its effects upon the thyroid and its probable liquid crystalline properties.

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Zusammenfassung In drei Untersuchungen (I, II, IV) wurden männliche, 115–150 Tage alte Ratten entweder einem 0,5–3 oder einem 3–30 Gauss ELF (0,5 Hz) rotierenden magnetischen Feld (RMF) für 5, 10 oder 26 Tage ausgesetzt. Die Ratten, die dem RMF für 10 und 26 Tage ausgesetzt waren, hatten einen signifikant (p < 0,05) höheren Wasserkonsum als die Kontrollgruppe. Auch die Tiere, die dem RMF für 5 Tage ausgesetzt waren, tranken mehr Wasser (p > 0,05). Die exponierten Tiere zeigten auch eine fortschreitende Abnahme (p < 0,02) des relativen Schilddrüsengewichts, dagegen eine grössere Gewichtszunahme (p < 0,001) bei bis zu 10 Tagen Exponierung und Zunahme des relativen Hodengewichts (p < 0,05) bei bis zu 26 Tagen Exponierung. Die Unterschiede in der Zahl der zirkulierenden Esinophilen und im relativen Nebennierengewicht waren nicht signifikant. In der vierten Untersuchung (III) mit Ratten die bei Versuchsbeginn 80 Tage alt waren fanden sich nach 21 Tagen Exponierung keine Unterschiede beim Vergleich mit der Kontrollgruppe. Die physiologischen Veränderungen durch ELF-Exponierung, werden über eine Wirkung auf der Schilddrüse und ihre möglichen flüssigen und kristallinen Eigenschaften erklärt.

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Resume Lors de trois essais (I, II et IV), on a exposé des rats mâles, adultes, âgés de 115 à 150 jours à un champ magnétique tournant (CMT) d'une très basse fréquence (FTB) — de 0,5 Hz — soit de 0,5 à 3 Gauss, soit de 3 à 30 gauss, et cela durant 5, 10 ou 26 jours. Les rats exposés au CMT durant 10 ou 26 jours ont bu plus d'eau que des rats de contrôle et cela de façon significative (p < 0,05). Le groupe exposé au CMT durant 5 jours seulement a aussi bu davantage d'eau, mais la différence n'est alors pas significative. Les rats exposés à la fois au CMT et au FTB ont aussi montré une diminution significative (p < 0,02) du poids relatif de la glande thyroïde, une augmentation du poids du corps (p< 0,001) jusqu'au 10ème jour d'exposition et une augmentation du poids des testicules pour 26 jours d'exposition. On n'a par contre pas constaté de différences significatives entre les groupes quant au nombre d'oesinophiles circulant dans le sang, ni quant au poids des glandes surrénales, bien que ces différences augmentassent avec la durée d'exposition. Dans un quatrième essai (III), des rats âgés de 80 jours au début d'une période d'exposition de 21 jours n'ont présenté aucune différence quant aux paramètres mentionnés ci-dessus. Les changements du comportement et de la physiologie dûs à une exposition au FTB sont discutés par rapport aux répercussions d'une telle exposition sur la thyroïde et ses propriétés liquides et cristalline.

     

The effects of the microwaves on E. coli cells depend on oxygen concentration and static magnetic field

The effects of non-thermal microwaves (MW), 10(-4) and 10(-10) W/cm(2), on conformation of nucleoids in E. coli cells were analyzed by the method of anomalous viscosity time dependence (AVTD). MW exposure was performed at different values of static magnetic field and concentration of oxygen, 8-90 microT, and 2.3-7.8 mg/l, respectively. It was shown, that slight changes in both static magnetic field and oxygen concentration result in significant changes of MW effects up to their disappearance. It was established, that changes in static magnetic field affected significantly the time kinetics of the MW effects. The obtained data provide further evidence for strong dependence of the effects of non-thermal microwaves on physical parameters of exposure and physiological factors. These dependences should be taken into account in replication studies. The obtained results encourage further investigation of possible modulation of non-thermal MW effects by additional electromagnetic fields.     

SOS mutator effect in E. coli mutants deficient in mismatch correction.

We have used bacteriophage lambda to characterize the mutator effect of the SOS response induced by u.v. irradiation of Escherichia coli. Mutagenesis of unirradiated phages grown in irradiated or unirradiated bacteria was detected by measuring forward mutagenesis in the immunity genes or reversion mutagenesis of an amber codon in the R gene. Relative to the wild-type, the SOS mutator effect was higher in E. coli mismatch correction-deficient mutants (mutH, mutL and mutS) and lower in an adenine methylation-deficient mutant ( dam3 ). We conclude that a large proportion of SOS-induced 'untargeted' mutations are removed by the methyl-directed mismatch correction system, which acts on newly synthesized DNA strands. The lower SOS mutator effect observed in E. coli dam mutants may be due to a selective killing of mismatch-bearing chromosomes resulting from undirected mismatch repair. The SOS mutator effect on undamaged lambda DNA, induced by u.v. irradiation of the host, appears to result from decreased fidelity of DNA synthesis.     

Rifampin-resistant replication of pBR322 derivatives in Escherichia coli cells induced for the SOS response.

Replication of plasmid pBR322 in Escherichia coli cells normally requires RNA synthesis and thus is sensitive to rifampin, an inhibitor of RNA polymerase. In cells induced for the SOS response, however, derivatives of pBR322 were found to replicate in the presence of rifampin. This rifampin-resistant replication of pBR322 requires the insertion of certain sequences of DNA. The replication depends on recF+ and DNA polymerase I.