Oxidative and SOS-response in Escherichia coli and their interference]

Interference between the oxidative and SOS responses in Escherichia coli was studied. The oxidative response involves both reactive oxygen scavenging system and DNA repair systems which are distinct from either the SOS or adaptive response to alkylating agents. The oxyR gene is a positive regulatory gene for the oxidative response and controls at least 9 proteins which are induced by treatment with H2O2. This gene is not a portion of the SOS regulon that involves at least 17 different genes in E. coli and controls the SOS response--another inducible and nonspecific repair activity. The SOS response was measured in E. coli PQ37 by means of a sfiA: :lacZ operon fusion according to "SOS Chromotest" in a completely automated system "Bioscreen C" (Labsystems, Finland). Our data have shown that: 1) H2O2 was a potent inducer of sfiA gene--one of the SOS genes; 2) there was strong negative effect of the oxidative response on the subsequent induction of the SOS response. In common with our previous findings it should be concluded that there is an interference between the SOS response--on the one hand, and the adaptive and oxidative responses--on the other. The nonspecific heat shock response is proposed to be a main key in these interferences.     

A comparative study of mutagenic and SOS-inducing activity of biphenyls, phenanthrenequinones and fluorenones

A total of 23 chemicals--biphenyls, phenanthrenequinones and fluorenones--were tested for mutagenicity towards Salmonella typhimurium strains TA1538, TA1535 and TA98. SOS-inducing activity of the same chemicals was studied in terms of the SOS-inducing potency in Escherichia coli PQ37, using an automated instrument controlled by a dedicated computer program for the SOS Chromotest. Of the 23 chemicals studied 14 induced His+ revertants in S. typhimurium TA1538 hisD305 (-1 frameshift); none induced His+ reversions in TA1535 (base-pair substitution). The mutagenicity of the chemicals in S. typhimurium TA98 (pKM 101) was lower than in TA1538. There was a close correlation between mutagenicity and SOS-inducing activity of fluorenones and phenanthrenequinones. None of the biphenyls tested induced SOS response and this property does not depend upon the mutagenic activity of the chemicals. SOS Chromotest is particularly valid in detecting chemicals which give rise to base-pair substitutions through SOS induction. If positive results are obtained, the Salmonella assay may be omitted. However, this test cannot replace the Ames test especially for the primary screening of mutagenicity of chemicals with unknown structure.     

In vitro potential genotoxic effects of surface drinking water treated with chlorine and alternative disinfectants

A battery of in vitro short-term tests revealing different genetic end-points was set up in order to study surface-water genotoxicity after disinfection with different biocides: sodium hypochlorite (NaClO), chlorine dioxide (ClO(2)) and peracetic acid (PAA). The surface water both before and after disinfection was concentrated by adsorption on C(18) silica cartridges and the concentrates containing non-volatile organics were divided into different portions for chemical analyses and biological assays. The following in vitro tests were conducted on the water concentrates dissolved in DMSO: the Salmonella mutagenicity assay with S. typhimurium strains TA98 and TA100; the SOS Chromotest with Escherichia coli, the Microtox and Mutatox assays with Vibrio fischeri; and gene conversion, point mutation and mitochondrial DNA mutability assays with D7 diploid Saccharomices cerevisiae strain. The results show that the SOS Chromotest and the yeast assays are highly sensitive in detecting genotoxicity. The surface-water extracts were very often toxic to most of the test organisms considered, partially masking their potential mutagenic activity. Therefore, the assays with E. coli and with S. cerevisiae are more likely to show a mutagenic effect because these organisms are generally less sensitive to most toxic compounds. Among the tested disinfectants, NaClO and ClO(2) increased water genotoxicity, whereas PAA was able to slightly reduce raw water activity. However, because the organic compounds in the lake water varied with the season of the year, the disinfection processes, at times, both increased and decreased the raw water activity.     

Genotoxic, mutagenic and recombinogenic effects of rauwolfia alkaloids

In the last decade, the possible correlation between the use of reserpine and rauwolfia drugs as antihypertensive agents and breast cancer incidence has been investigated. For the purpose of evaluating the mutagenic and genotoxic effects of these drugs, reserpine and ajmalicine were studied using the SOS Chromotest and the induction of gene conversion, crossing-over and reverse mutation in the yeast diploid strain XS2316. The results indicated a lack of genotoxic, mutagenic and recombinogenic effects.     

Genotoxic activity of 3-[3-phenyl-1,2,4-oxadiazol-5-yl] propionic acid and its peptidyl derivatives determined by Ames and SOS response tests

Compounds derived from 1,2,4-oxadiazole have being reported for their anti-inflamatory activity. However, those compounds should be devoid of any genotoxic side effect. In this work, the genotoxic activity of peptidomimetic moiety-containing 1,2,4-oxadiazoles derivatives was tested based on the Ames and SOS Chromotest. The results showed no mutagenic activity on the Ames test for 3-[3-phenyl-1,2,4-oxadiazol-5-yl] propionic acid (POPA) parental drug, but a weak SOS response induction on Chromotest. The chemical modifications reduced that response to a non-significative level, with l-phenylalanine peptidomimetic derivative being showing the lowest induction response. The results pointed out for the effectiveness of promoting chemical modifications of biological active compounds to increase its mode of action, showed in previous work, without increasing and even decreasing its DNA damage effect.     

Genotoxic activity of 3-[3-phenyl-1,2,4-oxadiazol-5-yl] propionic acid and its peptidyl derivatives determined by Ames and SOS response tests

Compounds derived from 1,2,4-oxadiazole have being reported for their anti-inflammatory activity. However, those compounds should be devoid of any genotoxic side effect. In this work, the genotoxic activity of peptidomimetic moiety-containing 1,2,4-oxadiazoles derivatives was tested based on the Ames and SOS Chromotest. The results showed no mutagenic activity on the Ames test for 3-[3-phenyl-1,2,4-oxadiazol-5-yl] propionic acid (POPA) parental drug, but a weak SOS response induction on Chromotest. The chemical modifications reduced that response to a non-significative level, with l-phenylalanine peptidomimetic derivative being showing the lowest induction response. The results pointed out for the effectiveness of promoting chemical modifications of biological active compounds to increase its mode of action, showed in previous work, without increasing and even decreasing its DNA damage effect.     

Using Short-Term Mutagenicity Tests for the Evaluation of Genotoxicity of Contaminated Soils

The study is aimed at evaluating the genotoxicity of contaminated soils using two bacterial mutagenicity assays — the Ames test and the SOS Chromotest. Initially, attention is directed at the method of extraction of soil samples by organic solvents. The detection of mutagenicity was dependent on the type of organic solvent. Dichloromethane (DCM) proved to be a better extraction agent than acetone because it is more effective for extracting mutagenic compounds. In the second part of our study, the possibilities of using bacterial mutagenicity assays for monitoring the course and effectiveness of bio-remediation of contaminated soils were ascertained. The results of an evaluation of the genotoxicity of a residue of polycyclic aromatic hydrocarbons (PAHs) that decompose with difficulty showed that a decrease in the concentration of detectable components need not always correspond to a total decrease of the mutagenic effect. Contaminants inducing SOS repair were degraded relatively quickly in soils, whereas it was found that mutagens inducing frameshift mutations persisted in samples.     

Genotoxic activity of two furan analogues of benzo[a]pyrene and their 2-nitro derivatives

We measured the genotoxic activities in two bacterial tests, the Salmonella/histidine assay (a reverse mutation assay) and the SOS Chromotest (an assay for SOS induction in E. coli), of two pairs of isomeric furan analogues of benzo[a]pyrene: pyreno[1,2-b]furan (R7490) and pyreno[2,1-b]furan (R7692) and their 2-nitro derivatives, 8-nitro-pyreno[1,2-b]furan (R7489) and 8-nitro-pyreno[2,1-b]furan (R7691). We found that: For all 4 compounds, the responses were correlated in the two tests. For the 2-nitro derivatives, R7489 and R7691, the responses were extremely high, reaching SOS-inducing potencies of 5.2 X 10(3) and 10(5)/nmole in the SOS Chromotest and mutagenic potencies of 6.3 X 10(4) and 3.7 X 10(7) revertants/nmole in the Salmonella/histidine assay (strain TA98), respectively; the responses were only slightly decreased in nitroreductase-deficient strains. The responses to the two pyrenofurans were increased in the presence of an "activating mixture" but were still lower than that to benzo[a]pyrene. In contrast to benzo[a]pyrene and pyreno[2,1-b]furan (R7692), pyreno[1,2-b]furan (R7490) also gave a response in the absence of an "activating mixture". (5) Compounds with the oxygen heteroatom within the "bay region" gave lower responses than their isomers with the oxygen heteroatom outside the "bay region".     

SOS system induction in Escherichia coli cells with distinct levels of ribonucleotide reductase activity.

The UV-mediated induction of recA and sfiA genes in Escherichia coli cells with distinct levels of dATP has been studied. Low levels of dATP were obtained by using either a temperature-sensitive ribonucleotide (RDP) reductase-deficient (nrdA) mutant or a wild-type strain treated with hydroxyurea. High pools of dATP were achieved by using a plasmid overproducing RDP reductase. The results obtained show that expression of the recA and sfiA genes was inhibited neither in the UV-irradiated nrdA mutant at 42 degrees C nor in the wild-type strain in the presence of hydroxyurea. Likewise, the increase of the dATP pool did not enhance recA and sfiA gene expression after UV irradiation. All these data suggest that the basal level of dATP is not a limiting factor in the process of induction of the SOS system in Escherichia coli.     

Absence of genotoxic activity of refined smoke flavor (RSF) in two bacterial short-term tests

The genotoxic activities of refined smoke flavor (RSF) produced in Poland and used in food processing were investigated in 2 bacterial short-term tests. Its mutagenic activity was examined in the Salmonella/histidine plate assay and its SOS-inducing capacity in the SOS Chromotest both without and with 'activation' by a rat liver homogenate. No genotoxic activity was detected using these 2 bacterial tests.     

Genotoxicity assessment of low-molecular weight interferon inducers by the SOS Chromotest

Tilorone and its aza-analogs, as well as CMA and its butyric analog (CNPA) were investigated as potential genotoxic agents by the SOS Chromotest. The SOS-inducing potency values (SOSIP) were 0.0033 and 0.0009 for SAF and vivakorfen, respectively, after activation with S9 fraction of mouse liver only. In contrast, an SOSIP value for tilorone of 0.0011 was observed in a non-activated assay. The SOSIPs of investigated compounds were low and comparable to the lowest values determined for other genotoxins. CMA and CNPA were not SOS inducers in any test system.     

Mutagenicity of 43 structurally related heterocyclic compounds and its relationship to their carcinogenicity.

43 heteropolycyclic compounds belonging to a homologous series were investigated for mutagenicity. The results are compared with carcinogenicity data obtained with the same batches of compounds under conditions identical for all of them. Mutagenicity was tested in the Ames test with Salmonella typhimurium strains TA1535, TA1537 and TA100 in the presence and absence of liver 10 000 g supernatant from rats treated with Aroclor 1254. Carcinogenicity was tested by injection of the compounds into subcutaneous tissue of XVIInc/Z mice. 18 test compounds showed carcinogenic activity, some strongly, others only weakly. Of these, 17 were detected as mutagens: one weak carcinogen did not revert the Salmonella strains. No quantitative correlation was observed between the extents of the mutagenic and the carcinogenic effects. Of the 25 substances that did not produce tumours, 13 showed mutagenicity (12 in the presence, 2 in the absence, of the liver homogenate). The mutagenic effects of these compounds were quantitatively similar to those of the compounds that produced tumours. The most sensitive strain of Salmonella typhimurium was TA100. It detected all 30 mutagens. TA98 was mutated by 25 compounds, TA1537 by 16 compounds. No mutagenic effects were seen with TA1535. Possible reasons for the high percentage of apparently "false positives" in the Ames test and the lack of a quantitative correlation between the potency of the mutagenic and carcinogenic effects are discussed. It is suggested that the complexity of the metabolism of these heterocyclic compounds may lead to critical differences in metabolism in mouse subcutaneous tissue in vivo and in liver homogenates from rats treated with Aroclor. Therefore the present study will be extended to life-long oral and intrahepatic carcinogenicity tests leading to a higher proportion of metabolism in the liver.     

Diesel emissions revisited: is the carcinogenicity due to a genotoxic mechanism?

The induction of recA, umuC and sfiA genes by quercetin was studied in the presence and in the absence of S9 mix. The inducing activity of quercetin is higher for sfiA than for recA and umuC genes in the absence of S9 mix. The putative genotoxic metabolites of quercetin produced by S9 mix display different inducing activities of the three SOS genes as compared to quercetin. The induction of sfiA gene is decreased by the presence of S9 mix, whereas an opposite effect was observed concerning umuC and recA. These data suggest that the error-prone repair pathway participates in mutagenesis by quercetin and its metabolites. Moreover, the type of DNA damage exerted by quercetin seems to be determined by its metabolic fate. The importance of testing for the induction of other SOS genes, together with sfiA, in the study of SOS functions as a genotoxic index is emphasized.     

Study of the genotoxic potential of 48 inorganic derivatives with the SOS chromotest

The genotoxic potential of 48 inorganic derivatives was studied using the bacterial colorimetric assay: the SOS Chromotest. Some of these compounds are known as carcinogens (As, CR(VI), Cd, Ni) or suspected carcinogens for human beings (Hg, Pb), others are known as non-carcinogens. Among these 48 derivatives, only the two Cr(VI) compounds and the Sn(II) compounds gave positive results.     

Induction of the SOS system in a dam-3 mutant: a diagnostic strain for chemicals causing DNA mismatches

We have developed a strain of E. coli in which expression of the SOS function sfiA, monitored by means of a sfiA::lacZ operon fusion, is efficiently triggered by the two base analogues 2-aminopurine and 5-bromo-2'-deoxyuridine. This strain resulted from introduction of a dam-3 mutation into a Uvr+, Rfa+ derivative of strain PQ37 used in the SOS chromotest, a bacterial colorimetric assay for genotoxins (Quillardet et al., 1982). The dam-3 mutation affects the mismatch correction system in E. coli. We show that the SOS-inducing capacity of a weak SOS inducer such as the alkylating agent ethyl methanesulfonate was also increased in the dam-3 strain. We provide evidence that the increase in SOS inducibility due to the dam-3 mutation is specific for compounds causing DNA mismatches and propose the use of the dam-3 derivative of PQ37 as a diagnostic strain for such agents. This diagnostic strain can be a useful addition to the SOS chromotest.     

Quantitative comparative mutagenesis in bacteria, mammalian cells, and animal-mediated assays A convenient way of estimating genotoxic activity in vivo?

The accumulation of environmental compounds which exhibit genotoxic properties in short-term assays and the increasing lag of time for obtaining confirmation or not in long-term animal mutagenicity and carcinogenicity tests, makes it necessary to develop alternative, rapid methodologies for estimating genotoxic activity in vivo. In the experimental approach used here, it was assumed that the genotoxic activity of foreign compounds in animals, and ultimately humans, is determined among others by exposure level, organ distribution of (DNA) dose, and genotoxic potency per unit of dose, and that knowledge about these 3 parameters may allow to rapidly determine the expected degree of genotoxicity in various organs of exposed animals. In view of the high degree of qualitative correlation between mutagenic activity of chemicals in bacteria and in cultured mammalian cells, and their mutagenic and carcinogenic properties in animals, and in order to be able to distinguish whether mutagenic potency differences were due to differences in (DNA) dose rather than other physiological factors, the results of mutagenicity tests obtained in the present experiments using bacteria and mammalian cells were compared on the basis of DNA dose rather than exposure concentrations, with the following questions in mind: Is there an absolute or a relative correlation between the mutagenic potencies of various ethylating agents in bacteria (E. coli K12) and in mammalian cells (V79 Chinese hamster) after treatment in standardized experiments, and can specific DNA adducts be made responsible for mutagenicity? Is the order of mutagenic potency of various ethylating agents observed in bacteria in vitro representative of the ranking of mutagenic potency found in vivo? Since the answer to this last question was negative, a further question addressed to was whether short-term in vivo assays could be developed for a rapid determination of the presence (and persistence) of genotoxic factors in various organs of mice treated with chemicals. In quantitative comparative mutagenesis experiments using E. coli K12 and Chinese hamster cells treated under standardized conditions in vitro with 5 ethylating agents, there was no indication of an absolute correlation between the number of induced mutants per unit of dose in the bacteria and the mammalian cells. The ranking of mutagenic potency was, however, identical in bacteria and mammalian cells, namely, ENNG greater than ENU greater than or equal to DES greater than DEN congruent to EMS, the mutagenic activity of DEN being dependent on the presence of mammalian liver preparations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Relationships between structures and mutagenic potencies of 16 heterocyclic nitrogen mustards (ICR compounds) in Salmonella typhimurium

16 heterocyclic nitrogen mustards (ICR compounds), which were synthesized for use as possible antitumor agents by Creech and coworkers, were tested for mutagenicity in Salmonella typhimurium strains TA1535, TA1536, TA1537, TA1538, TA98 and TA100. The compounds were incorporated into the top agar at 5 doses: 0.5, 1, 2.5, 5 and 10 micrograms/plate. All of the compounds were negative in TA1535 except ICR 449, which was positive in all 6 strains. The other 15 compounds were positive in the remaining strains with the following exceptions: ICR 371 and 355 were negative in TA100; ICR 445 was negative in TA98 and TA100; and ICR 360 was negative in TA1537, TA1538, TA98 and TA100. Good qualitative agreement was observed between the mutagenic and antitumor activities of the 16 compounds, and between the mutagenic and carcinogenic activities of the 5 compounds that have been tested for carcinogenicity by Peck and coworkers. However, no significant correlation was found between mutagenic potency in Salmonella and antitumor potency in mice for the 16 compounds. Also, for the 5 compounds that have been tested for carcinogenicity, no significant correlation was found between their mutagenic potency in Salmonella and their carcinogenic potency in mice. In Salmonella, the secondary (2 degrees) amines generally were more mutagenic than their tertiary (3 degrees) amine homologs, although the opposite result has been reported in certain eukaryotes. Relationships between structures and potencies for the different nuclei of the 16 ICR compounds are discussed, as are similarities and differences in strain sensitivities. We conclude that the Salmonella his reversion test is not a good predictor of the antitumor and carcinogenic potencies of these ICR compounds.     

Detection of mutagens in water-distribution systems after disinfection

This research examined the quality of water-before and after distribution-of four drinking-water production plants located in Northern Italy, two of which collected water from local aquifers and two from the River Po. A battery of genotoxicity assays for monitoring drinking-water was performed to assess the quality of the water produced by the treatment plants under study. Three different sampling stations were selected at each plant, one right at the outlet of the treatment plant and two along with the distribution pipelines. Raw river water was also sampled and analysed as a control. The water samples (500 l) were concentrated on silica C18 cartridges and the extracts were tested in in vitro mutagenicity assays (Salmonella/microsome assay with strains TA 98 and TA 100; SOS Chromotest with Escherichia coli strain PQ37); gene conversion, point mutation and mitochondrial DNA mutability assays with the diploid Saccharomyces cerevisiae strain D7 and a toxicity test using the bioluminescent bacterium Vibrio fischeri (Microtox). The Microtox test and the mitochondrial DNA mutability assay showed the greatest sensitivity towards toxic or mutagenic substances in the water extracts considered. The results show that this battery of short-term tests is applicable in the routine monitoring of drinking-water quality before and after distribution.     

Cytotoxicity and SOS-inducing ability of ethidium and photoactivable analogs on E. coli ethidium-bromide-sensitive (Ebs) strains

In a recently-characterized ethidium-bromide-sensitive E. coli strain, DNA appears to be much more accessible to DNA-binding agents. This strain therefore appears to be of interest for studying the mutagenic properties of chemicals. For this purpose, a series of ethidium-sensitive E. coli strains (Ebs) with normal and defective DNA-repair capacity was constructed and made lysogenic for lambda (sfiA::lacZ). These strains were used to study the cytotoxicity and SOS-inducing ability of ethidium and its two photoactivable analogs 8-azido- and 3,8-diazido-ethidium. When non-covalent DNA complexes are formed, these dyes elicit only a bacteriostatic effect in the Ebs strains, which is almost independent of the strain's DNA-repair capacity. The SOS system is not induced. When covalent DNA adducts are formed after photoactivation of ethidium azido analogs, the effects are quite different. The formation of about 5 DNA monoadducts per cell induces a lethal hit in the Ebs uvrB recA strain and measurable SOS induction in the Ebs uvrB (lambda (sfiA::lacZ) strain. The formation of more than 1000 DNA adducts in the Ebs strain with normal DNA-repair capacity does not induce any measurable cytotoxic effect.