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PubMed1996

Induction of SOS-independent mutations by benzo[a]pyrene treatment in Escherichia coli cells deficient in MutY or MutM DNA glycosylases: possible role of oxidative lesions.

The induction of SOS-independent mutations by exposure to benzo[a]pyrene (BaP) was screened in Escherichia coli strains lacking SOS mutagenesis proteins and deficient in MutY or MutM glycosylases, which prevent mutations by 8-hydroxyguanine (GO lesion). Mutagenicity assays, performed in the presence of S9 mix, indicated a great increase in the reversion of the trpE65 ochre mutation in both mutY and mutY mutM strains, whereas a lower increase was observed in a mutM strain. This mutability by BaP was observed in either uvr- or uvr+ strains. Moreover, it was increased when strains carried a deletion of the oxyR gene that abolished the OxyR response to oxidative stress, and reduced in the presence of the oxyR2 allele that rendered constitutive such response. It is suggested that SOS-independent mutations in cells treated with BaP arise from adenine/GO mispairs. The interaction of radical scavengers with BaP ultimate metabolites could cause oxidative stress capable of producing GO lesions. Strains lacking mutagenesis proteins and deficient in base excision repair systems, such as those dependent on MutY and MutM glycosylases, could be useful for screening the induction of SOS-independent mutations.

Adenosine Triphosphatases↗
PubMed1996

Nitro musks are cogenotoxicants by inducing toxifying enzymes in the rat.

In the present study, musk xylene (MX) and musk ketone (MK) were examined for their potency to induce toxifying enzymes in the liver of Sprague-Dawley rats, using an in vivo/in vitro model. After i.p. application of 10, 20 and 40 mg/day MX and MK over a period of 5 days, 9000 x g liver fractions (S9M) were used to study the toxification of a number of well-known pregenotoxicants in the SOS chromotest, i.e., benzo[a]pyrene (B[a]P), 2-aminoanthracene (2-AA), and aflatoxin B1 (AFB1). The genotoxic potencies of B[a]P, 2-AA and AFB1 in the presence of S9M were compared to those obtained in the presence of S9 fractions of untreated animals (S9O, negative control). S9M fractions derived from MK-treated rats showed an increased potency to toxify B[a]P, 2-AA and AFB1 in comparison to S9O fractions (for instance: TIP[toxifying induction potency] = 70 per nmol AFB1 using 10 mg MK treatment). In comparison, S9M fractions from MX-pretreated rats exhibited an increased genotoxicity only when using 2-AA (TIP = 0.04) and AFB1 (TIP = 61) as pregenotoxicants, but not when using B[a]P. To summarize the results, both MX and MK were strong inducers of toxifying liver enzymes. Therefore, these compounds seem to be cogenotoxicants for a number of well-known pregenotoxicants. Synergistic effects were found when using inducers of toxifying enzymes and pregenotoxicants in the in vivo/in vitro induction model.

Animals↗
PubMed1996

Effect of overexpression of E. coli 3-methyladenine-DNA glycosylase I (Tag) on survival and mutation induction in Salmonella typhimurium.

Salmonella typhimurium, compared to Escherichia coli, is deficient in an inducible glycosylase activity harbouring only constitutive glycosylase functions. 3-Methyladenine-DNA glycosylase I encoded by the E. coli tag gene is a constitutively expressed repair enzyme that primarily removes N3-methyladenine but also N3-methylguanine from DNA by glycosylic cleavage in the first step of the base excision repair. In order to investigate in vivo effect of the overexpressed glycosylase I activity on survival capacity and mutation induction in S. typhimurium, and thereby elucidate the significance of both 3-methylpurines in cellular sensitivity to methylating agents (e.g., DMS), we transformed four his- S. typhimurium strains with the plasmid pCY5 carrying the E. coli tag gene under the control of the lac promoter. Although the 3-methyladenine-DNA glycosylase activity in cells carrying pCY5 was only 10-fold higher on exposure to IPTG compared to the TA1535 control strain carrying pUC8, the overexpression of the Tag protein completely suppressed deficiency in an inducible glycosylase activity, rendering cells resistance to toxic effects of DMS. The suppression was not influenced by the nucleotide excision repair pathway since there was no difference in recovered survival among NER-proficient and NER-deficient strains. The yield of mutation induction in the reversion assay was decreased to the level of spontaneous (his-->his+) revertant colonies showing that in the overall population in overexpressed conditions in vivo 3-methyl-guanine, in addition to 3-methyladenine, must have been removed from DNA by the E. coli Tag protein and therefore accounts for the second most important cytotoxic lesion.

Adenine↗
PubMed1996

Influence of the uvr-dependent nucleotide excision repair on DNA adducts formation and mutagenic spectrum of a potent genotoxic agent: 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000).

The influence of the uvr-dependent excision repair system on the lethal action, mutagenic specificity, SOS induction and DNA adducts formation of 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000), a potent genotoxic nitrofuran, were examined in Escherichia coli. Binding measurements of 3H-labelled R7000 to DNA indicated that R7000-DNA adducts can be removed by excision repair soon after the action of the chemical: 50% of the DNA adducts were removed within 10 min of treatment. After 1 h of incubation the level of excision reached 70%. This result was confirmed using the postlabelling technique. We found that R7000 yielded at least 10 different DNA adducts. Each of the adducts detected could be removed by excision repair. The rates of excision appeared different from one to the other. In addition, using a lacZ reversion system that is able to detect each type of base substitution mutations [1], we found that in uvrA bacteria deficient in excision repair, R7000 can induce 5 out of the 6 possible mutational events: GC-->TA, AT-->TA, GC-->CG, AT-->CG and GC-->AT. The transition AT-->GC was not observed. Only 3 transversions: GC-->TA, AT-->TA and GC-->CG could be detected in repair proficient uvr+ bacteria. The differences between the mutagenic spectra obtained in either uvr+ bacteria or uvrA mutants indicate that some potentially mutagenic DNA adducts induced by R7000 can be removed by excision repair, thus lowering the mutagenic potency of the chemical and modifying the mutagenic spectrum detected.

Bacterial Proteins↗
PubMed1987

Reinitiation at the lambda DNA origin accompanies the host SOS response.

Abnormal reinitiation of replication from lambda origins has previously been found during infection in the presence of caffeine or cis-diamminedichloroplatinum II (cis-Pt) or when lambda infects a P2 lysogen. It was further shown that the reinitiations arising from cis-Pt treatment took place during the SOS response induced by the template damage caused by the drug. It is now shown that SOS induction by uv irradiation of the host also results in reinitiation events and that it is the SOS response itself rather than some other direct effect of the damaged host template that is responsible for the phenomenon. Parental sections of lambda replicative intermediates can supercoil, whereas daughter segments cannot. To explain the control that prevents reinitiation, it is proposed that normally the origin sequence has to be under superhelical tension to be a suitable substrate for the initiating machinery; once a round is in progress, the daughter origin sequences would not be under such tension and would therefore be inactive. It is shown that in an SOS environment the proposed requirement for a superhelical origin sequence is relaxed and consequently the control against reinitiation lost. Under such conditions, primary growing points that have encountered template lesions terminate and a new wave of replication initiates.

Bacterial Proteins↗
PubMed1989

The mutagenicity of refuse leachate from a municipal incinerator.

The mutagenicity of refuse leachate from a municipal incinerator was studied by liquid rec-assay and Ames assay. Volatile components were found to be negative, and nonvolatile components positive, in the Ames assay and the leachate was found to have DNA-damaging capacity in the liquid rec-assay with S-9 mix. PAHs derived from tobacco ash and carbonyl compounds generated by the putrefaction of foods were confirmed to be main contributors to DNA-damaging capacity and mutagenicity in refuse leachate.

Animals↗
PubMed1990

Regulation in repressor inactivation by RecA protein.

Treatments that damage DNA or inhibit DNA synthesis in E. coli induce the expression of a set of functions called SOS functions that are involved in DNA repair, mutagenesis, arrest of cell division and prophage induction. Induction of SOS functions is triggered by inactivation of the LexA repressor or a phage repressor. Inactivation of these repressors results from their cleavage by the E. coli RecA protein in the presence of single-stranded DNA and a nucleoside triphosphate. We found that these cleavage reactions are controlled by two mechanisms in vitro: one is through the structural change of the RecA protein in the ternary complex, RecA-ssDNA-ATP-gamma-S. The active ternary complex is formed by binding of ATP-gamma-S to a complex of RecA protein and ssDNA. On the other hand, when the RecA protein binds to ATP-gamma-S prior to its binding to ssDNA, the resulting complex has no or only very weak cleavage activity toward the repressor. This structural change is negatively controlled by its C-terminal part. The loss of the 25 amino acid residues from the C-terminal leads the RecA protein to stable binding to dsDNA as well as ssDNA, and the protein takes the activated form for the repressor cleavage constitutively. The other mechanism is through the structural change of the repressor. The cleavage reaction of a phi 80cI repressor is greatly stimulated by the presence of d(G-G), and d(G-G) stimulates the cleavage by binding to the C-terminal half of the phi 80cI repressor. Moreover, the C-terminal fragment of the cleaved products of the 80cI repressor was able to cleave a phi 80cI-lambda chimeric repressor. These results strongly suggested that the active site of the repressor cleavage was located in the C-terminal domain of the repressor and that the C-terminal fragment produced by the cleavage could cleave the repressor.

DNA Damage↗
PubMed1981

Regulation of SOS functions: purification of E. coli LexA protein and determination of its specific site cleaved by the RecA protein.

The LexA protein of Escherichia coli was purified to more than 96% purity from cells harboring a recombinant plasmid carrying the lexA gene with the lacZ promoter sequence. The amino acid composition of the LexA protein and its amino-terminal sequence were analyzed. The results are in agreement with the prediction from the nucleotide sequence of the lexA gene. The LexA protein is cleaved into two polypeptides by E. coli RecA protein in the presence of ATP and single-stranded DNA. The site of the specific cleavage was determined by analyzing amino acid sequences of the cleaved products at the amino and carboxyl termini. The cleavage of the LexA protein by the RecA protein was found to occur at a single site between Ala84 and Gly85.

Bacterial Proteins↗
PubMed1989

A specific class of IS10 transposase mutants are blocked for target site interactions and promote formation of an excised transposon fragment.

We report the identification and characterization of a class of IS10 transposase mutants that carry out only some of the steps required for transposition. These mutants were identified among transposition-defective mutants as a specific subclass that retains the wild-type ability to induce SOS functions in the presence of transposon ends. Mutants of this class successfully promote excision of the element from its donor site, but do not promote transfer of the transposon sequences to a target site. SOS induction presumably results from the degradation of the donor site. Uniquely among transposition-defective mutants, SOS+ Tnsp- mutants promote the formation of a new product, the excised transposon fragment (ETF), which consists of the transposon excised from the original donor molecule by double-strand breaks at the transposon ends. SOS+ Tnsp- mutants identified thus far define two patches of amino acids that might correspond to regions of different function. A single additional mutation maps within a region that is highly conserved among IS element transposases. The existence of SOS+ Tnsp- mutants and the structure of the ETF provide strong support for the previously proposed nonreplicative model of Tn10/IS10 transposition.

Alleles↗
PubMed1989

Effect of induction of SOS response on expression of pBR322 genes and on plasmid copy number.

Several lines of evidence are presented that indicate that the level of tetracycline resistance of Esherichia coli strains harboring plasmid pBR322 varies according to whether the SOS system of the host bacteria has been induced. These include use of strains in which the SOS system is expressed constitutively (lexA def.), is thermoinducible (recA441) or noninducible (lexA ind-), or is highly repressed (multiple copies of lexA+). Similar induction was observed with the product of another plasmid gene, beta-lactamase. The amounts of extractable plasmid DNA were also increased by SOS induction, and we propose that the SOS-induced increases in levels of tetracycline resistance and beta-lactamase activity are due to an increased plasmid copy number.

Alleles↗
PubMedDate not supplied

Application of a semi-automated SOS chromotest for measuring genotoxicities of complex environmental mixtures containing polycyclic aromatic hydrocarbons.

Soxhlet-extracted samples of standard reference materials (SRMs) 1649 (PAR1: urban dust/organics) and 1650 (PAR2: diesel particulate matter) from the U.S. Institute of Standards and Technology were tested for induction of SOS functions using a semi-automated version of the SOS chromotest with Escherichia coli PQ37. Concentrations of 10 polycyclic aromatic hydrocarbons in the extracts were determined using reversed-phase HPLC. Only the diesel particulate matter (PAR2) extracts expressed SOS induction activity, which decreased when metabolic activation was used. Mutagenic PAH compounds (e.g., chrysene) were found in higher concentrations in the PAR2 extracts than in the PAR1 extracts but this could not explain the genotoxicity while it was mainly exhibited without metabolic activation. The direct genotoxic activity of the diesel particulate matter sample PAR2 is probably caused by nitroaromatic compounds; this was also supported by parallel studies with the Ames/Salmonella assay.

Air Pollutants↗
PubMed1993

The SOS chromotest: a review.

The SOS chromotest is reviewed through over 100 publications corresponding to the testing of 751 chemicals. 404 (54%) of these chemicals present a genotoxic activity detectable in the SOS chromotest. Their SOS inducing potencies span more than 8 orders of magnitude. For 452 compounds, the results obtained in the SOS chromotest could be compared to those obtained in the Ames test. It was found that 373 (82%) of these compounds give similar responses in both tests (236 positive and 137 negative responses). Thus the discrepancies between both tests concern 79 compounds (18%). A case by case analysis shows that many of these compounds are at the same time very weak SOS inducers and very weak mutagens. Thus we think that, most of the time, the discrepancies between the two tests may be accounted for by differences in the interpretation of the results rather than by the experimental results themselves. However, there are some compounds which are clearly SOS inducers but devoid of mutagenic activity in the Ames test (such as quinoline-1-oxide) and to a larger extent, clearly mutagenic compounds which do not induce the SOS response in the SOS chromotest (such as benzidine, cyclophosphamide, acridines, ethidium bromide). We also analyzed the correlation between SOS induction, mutagenesis and carcinogenesis according to the classification of Lewis. For 65 confirmed carcinogens (class 1), the sensitivity, i.e., the capacity to identify carcinogens, was 62% with the SOS chromotest and 77% with the Ames test. For 44 suspected carcinogens (class 2), the sensitivity was 66% with the SOS chromotest and 68% with the Ames test. Thus, we confirmed previous observations made on 83 compounds that there is a close correlation between the results given by both bacterial tests. The capacity of the Ames test to identify carcinogens is higher than that of the SOS chromotest. However, because the number of false positive compounds was lower in the SOS chromotest, the specificity, i.e., the capacity to discriminate between carcinogens and non-carcinogens of the SOS chromotest, appeared higher than that of the Ames test. Thus, the results of the SOS chromotest and of the Ames test can complement each other. The SOS chromotest is one of the most rapid and simple short-term test for genotoxins and is easily adaptable to various conditions, so that it could be used as an early--perhaps the earliest--test in a battery.

Animals↗
PubMed1993

Some aspects of EMS-induced mutagenesis in Escherichia coli.

AB2497 and its mutS and umuDC derivatives were EMS-treated at the stationary phase and specificity of mutation measured. It was found that: (i) in mutS+ cells EMS induces predominantly GC-->AT transitions (by supB or supE(oc) formation) and in mutS- cells mainly AT-->TA transversions (by supL(NG) formation); (ii) transversions of AT-->TA are umuDC-dependent and mutational specificity is biased towards AT-->GC transitions in mutS- umuDC- strains. When mutS- umuDC- cells were transfected with plasmids bearing umuD'C or umuDC genes, mutational specificity was again biased towards AT-->TA transversions; (iii) experiments with bacteria bearing umuC::lacZ or recA::lacZ fusions suggest that processing of UmuD-->UmuD' might be poorer in EMS-treated mutS- than in mutS+ cells.

Bacterial Proteins↗
PubMed1986

Kinetic determination of enzymatic activity and modification of the metabolic activation system in the SOS chromotest.

The "SOS Chromotest" has recently been introduced by P. Quillardet et al. (1982; Quillardet and Hofnung, 1985), who use strain PQ37 of Escherichia coli K12 to test for genotoxicity. We have modified the procedure in order to optimize the determination of beta-galactosidase and alkaline phosphatase activities, and, where possible, to allow measurements to be made automatically. Kinetic determination is quicker, more sensitive and avoids interference by coloured compounds. Modification of the metabolic activation system increases the sensitivity of the test for progenotoxicity.

Alkaline Phosphatase↗
PubMed1988

Evaluation of the SOS chromotest.

In the present investigation, the SOS chromotest with E. coli PQ37 was evaluated. The potential to identify different kinds of bacterial mutagens was examined. 124 chemicals of different chemical classes were tested. Their responses in the SOS chromotests were compared to reported test results obtained with the Ames test.

DNA Repair↗
PubMed1989

Detection of ionizing radiations with the SOS Chromotest, a bacterial short-term test for genotoxic agents.

The effects of 3 types of ionizing radiation, gamma-rays, neutrons and accelerated alpha-particles, were examined using the SOS Chromotest, a bacterial colorimetric assay for genotoxic agents based on the measurement of the SOS response in Escherichia coli. The SOS Chromotest appeared to be a sensitive and simple assay to detect quantitatively these radiations as well as their biological effects. The range of adsorbed doses for which induction was observed was similar for the 3 types of radiation, the minimum inducing doses being in the order of 2.5-5 Gy. We discuss the possible use of these observations to study the molecular action of radiations and to compare their genotoxic effects with those of chemicals.

DNA Repair↗
PubMed1991

Sources of variability of the Escherichia coli PQ37 genotoxicity assay (SOS chromotest).

To determine the variability in test results obtained with the Escherichia coli PQ37 genotoxicity assay (SOS chromotest) when varying the test protocol, we examined the influences of sodium dodecylsulfate (SDS) concentrations, of buffer pH and composition on the enzyme assays, the effects of E. coli PQ37 density and culture conditions on the expression and/or determination of alkaline phosphatase (ap) and beta-galactosidase (beta-g) activities, the calculated induction factors (IF) and the SOS-inducing potentials (SOSIP). Initially, we used 0-190 ng (0-1 nmole) 4-nitroquinoline-1-oxide (4-NQO) as a reference compound for the standard procedure in the absence of metabolic activation. Subsequently, to evaluate the results of protocol variations we examined several mutagenic compounds of differing chemical classes using both the standard and a modified assay procedure. We observed the highest enzyme activities using 1 mg SDS per tube and calibrating the ap buffer to pH 8.05 and the beta-g buffer to pH 7.75. The longer the incubation period, the higher the enzyme activities. However, with respect to IF and SOSIP there is no reason to incubate in excess of 90 min. We found no significant differences in the IF and SOSIP values when varying substrate conversion times. There was, however, a definite decrease in beta-g activity when extended substrate incubation times were used. Higher enzyme activities are obtained when the bacterial count is increased. Using lower bacterial counts the enzyme activities decreased, but the sensitivity of E. coli towards genotoxic compounds increased.

4-Nitroquinoline-1-oxide↗
PubMed1992

A sensitive umu test system for the detection of mutagenic nitroarenes in Salmonella typhimurium NM1011 having a high nitroreductase activity.

A sensitive umu test system for the detection of mutagenic nitroarenes has been developed using a new tester strain Salmonella typhimurium NM1011 having a high nitroreductase activity. The new strain was constructed by subcloning the bacterial nitroreductase gene into a plasmid pACYC184 and introducing the plasmid into the original strain S. typhimurium TA1535/pSK1002 harboring a fusion gene umuC'-'lacZ (pSK1002). Thus, the tester strain enabled us to monitor the genotoxic activities of various nitroarene compounds by measuring the beta-galactosidase activity in the cells. The sensitivity of strain NM1011 was compared with that of the parent tester strain S. typhimurium TA1535/pSK1002 or a nitroreductase-deficient strain S. typhimurium NM1000 with respect to the induction of umuC gene expression by 17 mutagenic nitroarenes. The newly developed strain with high nitroreductase activity had about 3 times higher nitrofurazone-reductase activity than the parent strain and was highly sensitive to the compounds 2-nitrofluorene, 1-nitronaphthalene, 2-nitronaphthalene, 1-nitropyrene, m-dinitrobenzene, 4,4'-dinitrobiphenyl, 3-nitrofluoranthene, 3,7-dinitrofluoranthene, 3,9-dinitrofluoranthene, 5-nitroacenaphthene and 2,4-dinitrotoluene. By contrast, the enzyme-deficient strain did not show any considerable response to 2-nitrofluorene, m-dinitrobenzene, 1-nitronaphthalene, 2-nitronaphthalene, 1-nitropyrene, 4,4'-dinitrobiphenyl, 3-nitrofluoranthene, 3,7-dinitrofluoranthene, 2,4-dinitrotoluene and 5-nitroacenaphthene. These results suggest that the newly developed tester strain with high nitroreductase activity is very useful for the detection of potent mutagenic nitroarene compounds.

Cloning, Molecular↗