Mutagenic activity of carcinogenic and noncarcinogenic nitrofurans and of urine of rats fed these compounds

The nitrofurans, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT), nitrofurantoin, 5-nitro-2-furoic acid, 5-nitro-2-furamidoxime, 5-nitrofurfurylidene diacetate and the urine of rats fed these compounds, were assayed for mutagenic activity in Salmonella typhimurium strains TA100 and TA100FR1. All the nitrofurans were mutagenic in the order: AF-2 and FANFT > nitrofurantoin > 5-nitro-2-furamidoxime > 5-nitrofurfurylidene diacetate > 5-nitro-2-furoic acid. Strain TA100 was more sensitive than TA100FR1 to the mutagenic influence of these nitrofurans. Only the urine of rats fed AF-2, FANFT and nitrofurantoin had mutagenic activity. Again, TA100 was more sensitive than TA100FR1. The mutagenicity of the urine was not increased by treatment with β-glucuronidase. AF-2, 2-amino-4-(5-nitro-2-furyl)thiazole (deformylated product of FANFT) and nitrofurantoin were excreted in the urine of rats fed these compounds; whereas the other nitrofurans were not excreted.     

Metabolic activation of dimethylnitrosamine and diethyl-nitrosamine to mutagens

Dimethylnitrosamine (DMN) and diethylnitrosamine (DEN) are not mutagenic by themselves, but they can be converted by mammalian enzymes to highly mutagenic products. As indicators for mutagenic activity, Neurospora crassa and Salmonella typhimurium were used. The ad-3 forward-mutation system was used to detect specific locus mutations; mutants in this system can range from multi-locus deletions to leaky mutations. The induction of mutations in S. typhimurium is detected as induction of histidine revertants of the histidine-requiring strain G₄6. The activation of DMN is microsomal, inhibited by SKF 525-A, and requires co-factors. The activating enzyme is induced in mice by pretreatment with phenobarbital, 3-methylcholanthrene and butylated hydroxytoluene. The mutagenic activity of the reaction products is directly correlated with the metabolic formation of formaldehyde with and without induction by 3-methylcholanthrene and across strains of mine. Formaldehyde does not contribute to the mutagenic activity of the reaction products. It is clear from the data that the reversion sites in G₄6 are more sensitive than the ad-3 loci of Neurospora crassa to the mutagenic action of DMN metabolites formed by mammalian liver. The microsomal assay is a few orders of magnitude more sensitive than the intraperitoneal host-mediated assay, and the intrahepatic host-mediated assay is a few orders of magnitude more sensitive than the in vitro microsomal system.     

Mutants of the phototrophic bacteria Rhodobacter sphaeroides sensitive to the mutagenic action of long-wave ultraviolet light

The mutagenic action of near ultraviolet (NUV, greater than or equal to 280) nm) on purple phototrophic soil bacteria Rhodobacter sphaeroides: wild strain 2R and 12 mutants obtained earlier sensitive to UV derivates (UVS) was investigated. The mutagenic action of NUV was measured by induction of resistance to tetracycline (Tet) and nalidixic acid (Nal) and reversion of pigment mutants to wild-type phenotype. The NUV light induces the mutations of resistance to Nal and Tet in wild-type strain 2R; the UVS mutants differed greatly in their NUV-induced mutability. Three UVS mutants were characterized by greatly increased mutability in all analysed loci; slight mutability was found in seven mutants. On the basis of the data obtained it has been concluded that the UVS mutants R. sphaeroides can be used as test organisms in estimation of mutagenic activity of NUV. The molecular mechanisms and genetic control of NUV-induced mutagenesis are discussed.     

The Activity of Pipemidic Acid and Piromidic Acid against Escherichia coli In Static Culture and in Conditions Simulating the Treatment of Bacterial Cystitis

The activity of the new pyridopyrimidine compounds, pipemidic and piromidic acids, against Escherichia coli was compared with that of nalidixic acid in vitro . In a static turbidimetric system nalidixic and pipemidic acids were found to be more active than piromidic acid when tested against sensitive E. coli strains, but pipemidic acid was the most active of the three compounds against nalidixic acid-resistant clinical isolates. When tested in an in vitro model designed to mimic the conditions in which bacteria and drug interact in the treatment of bacterial cystitis, all three drugs were found to be able to suppress bacterial growth for long periods, but a high, sustained drug level was necessary in order to prevent the emergence of resistant variants.     

Effect of Nalidixic Acid and Hydroxyurea on Division Ability of Escherichia coli fil+ and lon− Strains

Short periods of incubation in medium containing nalidixic acid or hydroxyurea, followed by a return to normal growth conditions, induced filament formation in Escherichia coli B (fil(+)) and AB1899NM (lon(-)) but not in B/r (fil(-)) and AB1157 (lon(+)). These drugs reversibly stopped deoxyribonucleic acid (DNA) synthesis with little or no effect on ribonucleic acid (RNA) synthesis or mass increase. The initial imbalance caused by incubation in these drugs was the same for B and B/r as was macromolecular synthesis following a return to normal growth conditions. DNA degradation caused by nalidixic acid was measured and found to be the same for B and B/r. Hydroxyurea caused no DNA degradation in these two strains. Survival curves as determined under various conditions by colony formation suggested that the property of filament formation was responsible for the extrasensitivity of fil(+) and lon(-) strains to either nalidixic acid or hydroxyurea. E. coli B was more sensitive to either drug than was B/r or B(s-1). Pantoyl lactone or liquid holding treatment aided division and colony formation of nalidixic acid-treated B but had no effect on B/r. Likewise, the filament-former AB1899NM was more sensitive to nalidixic acid than was the non-filament-former AB1157. The sensitivity of B/r and B(s-1) to nalidixic acid was nearly the same except at longer times in nalidixic acid, when B(s-1) appeared more resistant. Even though nalidixic acid, hydroxyurea, and ultraviolet light may produce quite different molecular alterations in E. coli, they all cause a metabolic imbalance resulting in a lowered ratio of DNA to RNA and protein. We propose that it is this imbalance per se rather than any specific primary chemical or photochemical alterations which leads to filament formation by some genetically susceptible bacterial strains such as lon(-) and fil(+).     

The toxicities and mutagenic properties of ethylidene gyromitrin and N-methylhydrazine with Escherichia coli as test organism

The toxicities and mutagenic properties of ethylidene gyromitrin (acetaldehyde N-methyl-N-formylhydrazone), the main poisonous substance of false morel (Gyromitra esculenta Pers. Fr.), and N-methylhydrazine, the possible metabolic end-product of ethylidene gyromitrin, were studied by microbial tests. N-methylhydrazine was strongly bacteriocidic against Escherichia coli, whereas ethylidene gyromitrin had no detectable effect on this bacterium. Repair-deficient E. coli strains were more sensitive to N-methylhydrazine than were isogenic strains with normal repair capacities. In the reversion tests, N-methylhydrazine increased the reversion frequency, whereas ethylidene gyromitrin caused no detectable change on the reversion rate.     

Mutagenicity of 1-fluoro-2,4-dinitrobenzene is affected by bacterial glutathione

Recently we have shown that Salmonella typhimurium tester strains have high levels of the tripeptide glutathione (GSH) and activity of GSH S-transferases (Summer et al., 1979). In continuation of the GSH-dependent suppression of mutagenicity of 1-chloro-2,4-dinitrobenzene in presence of S9 fraction (Summer et al., 1979), this paper is focused on the GSH-dependent detoxifying capacity of the bacterial tester strains. 1-Fluoro-2,4-dinitrobenzene (FDNB), an electrophilic agent, which is used to identify terminal amino acids in proteins (Sanger reagent), readily reacts with GSH leading to a dose-dependent depletion of bacterial GSH. Additionally, FDNB is a strong mutagen for Salmonella typhimurium TA100, TA1538 and TA98 without metabolic activation.Presumably owing to conjugation with bacterial GSH, FDNB in concentrations which were lower or equal to those of bacterial GSH were found to be not mutagenic. Accordingly, increasing amounts of bacteria in the test system require increasing amounts of FDNB for expression of mutagenicity.     

A new Salmonella tester strain,TA97, for the detection of frameshift mutagens: A run of cytosines as a mutational hot-spot

We have developed a new Salmonella tester strain, TA97, for use in the Salmonella/microsome mutagenicity test. DNA sequencing has shown that this strain contains and added cytosine, resulting in a run of six cytosines at the mutated site in the histidine D gene. Its mutagenic specificity is similar to that of the frameshift mutagen tester strain, TA1537, which also contains an added cytosine in a run of cytosines and is currently among the five standard tester strains used for general mutagen screening. We assessed the mutagenic potency of 21 frameshift mutagens for TA1537 and TA97. TA97 was considerably more sensitive than TA1537 to reversion by these frameshift mutagens. In addition, one agent, PR toxin (from Penicillium roqueforti), which was not detected by any of the previously existing standard tester strains, did revert TA97; and two substituted aryl-alkyl triazenes, which had not been reported previously to be frameshift mutagens, were mutagenic in this new tester strain. We suggest that TA1537 be replaced by TA97 for general screening of mutagenicity.     

Effects of Rice Seed Surface Sterilization with Hypochlorite on Inoculated Burkholderia vietnamiensis

When a combination of hydrogen peroxide and hypochlorite was used to surface sterilize rice seeds, a 10²- to 10⁴-fold decrease in CFU was observed during the first 15 h after inoculation of the rice rhizosphere organism Burkholderia vietnamiensis TVV75. This artifact could not be eliminated simply by rinsing the seeds, even thoroughly, with sterile distilled water. When growth resumed, a significant increase in the frequency of rifampin- and nalidixic acid-resistant mutants in the population was observed compared to the control without seeds. This phenomenon was a specific effect of hypochlorite; it was not observed with hydrogen peroxide alone. It was also not observed when the effect of hypochlorite was counteracted by sodium thiosulfate. We hypothesized that the hypochlorite used for disinfection reacted with the rice seed surface, forming a chlorine cover which was not removed by rinsing and generated mutagenic chloramines. We studied a set of rifampin- and nalidixic acid-resistant mutants obtained after seed surface sterilization. The corresponding rpoB and gyrA genes were amplified and sequenced to characterize the induced mutations. The mutations in five of seven nalidixic acid-resistant mutants and all of the rifampin-resistant mutants studied were found to correspond to single amino acid substitutions. Hypochlorite surface sterilization can thus be a source of artifacts when the initial bacterial colonization of a plant is studied.     

Mutagenicity of 2-methylpropene (isobutene) and its epoxide in a modified Salmonella assay for volatile compounds.

The mutagenic properties of 2-methylpropene (MP) and 2-methyl-1,2- epoxypropane (MEP) were investigated in the Salmonella assay. A simple exposure system, consisting of gastight tissue culture flasks, was used. This method has the advantage that the volatile test chemical is present during the entire incubation period and that several concentrations of the investigated compound can be tested on a single day. MP is not mutagenic in strains TA100, TA102 and TA1535, and in the latter strain not even in the presence of metabolizing S9 mix. MEP is mutagenic in all the strains tested, as demonstrated by a clear dose-response relationship. Strain TA1535 seems to be most sensitive to MEP compared with the other bacterial strains studied. For this strain, the mutagenic activity of MEP decreased significantly in the presence of S9 mix, compatible with the epoxide being inactivated by epoxide hydrolase and by glutathione S-transferase, as reported previously. From the present study it can be concluded that the parent compound MP is not mutagenic, but that its primary metabolite MEP is a mutagenic substance. However, very high concentrations are necessary to induce a mutagenic effect and the epoxide is efficiently detoxified by different liver enzymes.     

N(4)-amino-and N(4)-hydroxycytosines as base analogue mutagens

N(4)-Aminocytosine [N(4)NH₂C] and N(4)-amino-2′-deoxycytidine [N(4)NH₂dC] are highly mutagenic for Escherichia coli and phage φ 80 but not for T₄. There is some evidence that they are incorporated into the φ 80 DNA but $\text{[}{}^{14}\text{C}\text{]-N(4)}\text{NH}{}_2\text{C}$ could not be detected in the bacterial DNA.N(4)-Hydroxy-5,6-dihydro-6-hydroxylaminodeoxycytidine (di-NHOH-dC) is mutagenic for φ 80 and E. coli, but N(4)-hydroxydeoxycytidine [N(4)OH-dC] only has a strong inactivating effect.     

Induction of Excessive Deoxyribonucleic Acid Synthesis in Escherichia coli by Nalidixic Acid

Prior treatment of Escherichia coli with nalidixic acid in nutritionally complete medium altered the subsequent pattern of deoxyribonucleic acid (DNA) synthesis normally observed in nutritionally deficient medium. Transfer of E. coli 15 TAU to an amino acid- and pyrimidine-deficient medium usually resulted in a 40 to 50% increase in DNA content. Previous treatment with nalidixic acid caused a 200 to 300% increase in DNA content under these conditions. The extent of this DNA synthesis depended on the duration of prior exposure to nalidixic acid. The maximal rate of synthesis was obtained after a 40- to 60-min exposure to nalidixic acid and was two to three times that of the control. The induction of this excessive DNA synthesis was prevented by chloramphenicol or phenethyl alcohol, but the synthesis of this DNA was only partially sensitive to these agents. With E. coli TAU-bar, the rate of DNA synthesis, after removal of nalidixic acid, was similar to that of E. coli 15 TAU, but the maximal amount of DNA synthesized was 180 to 185% of that initially present. Cesium chloride density gradient analysis demonstrated that DNA synthesis after removal of nalidixic acid occurs by a semiconservative mode of replication. The density distribution of this DNA was similar to that obtained after thymine starvation. These results suggest that nalidixic acid treatment may induce additional sites for DNA synthesis in E.coli.     

Phenotypic characteristics of thermotolerantCampylobacter from human and animal sources

Five reference strains and 314 field strains ofCampylobacter growing at 42°C, but not at 25°C, were characterized by tests of hippurate hydrolysis and sensitivity to nalidixic acid, to metronidazole, and to 2,3,5-triphenyltetrazolium chloride (TTC). All strains but seven were TTC-resistant by the disc test used. Of 168 human isolates, 87% hydrolyzed hippurate; three of these strains were nalidixic acid-resistant. Also hippurate-positive were all 23 strains from ovine abortion, 79% of 43 avian strains, two of six bovine isolates, and one of two strains of equine origin. Seventy-two porcine strains were all hippurate-negative. Metronidazole sensitivity was found to be a variable property in hippurate-positive strains, although present in all but four hippurate-negative strains. A group of eight nalidixic acid-resistant, hippurate-negative isolates of porcine and bovine origin probably represent a new group ofCampylobacter.     

Bactericidal effect of 5-azacytidine on Escherichia coli carrying EcoRII restriction-modification enzymes

5-Azacytidine was found to be bactericidal to Escherichia coli carrying plasmids specifying EcoRII restriction-modification systems, but not to the same strains lacking these plasmids. Of other base analogs tested, only 5(beta-D-ribofuranosyl)isocytidine had similar, although weaker, effects. Plasmids that had lost the EcoRII restriction-modification system did not confer sensitivity to 5-azacytidine. Mutants defective in the restriction function remained sensitive to the toxic effects of the drug; however, a mutant defective in the modification function lost most of the sensitivity to 5-azacytidine. For the bactericidal effect to be seen, the cells had to be growing; cells in the stationary phase of growth were not killed by the drug. The drug inhibited the methylase enzyme, and an inhibitor of the enzyme could be detected in vitro in extracts of cells that had been treated with 5-azacytidine. This nalidixic acid inhibited its formation. Coumermycin but not nalidixic acid antagonized the bactericidal effect of the drug; however, coumermycin was more effective in preventing the inhibition of the methylase by 5-azacytidine than was nalidixic acid.     

Kinetics of induction and decay of error-prone DNA repair activity in Escherichia coli after treatment with nalidixic acid

Inducible error-prone DNA repair activity was detected by infecting nalidixic acid-pretreated E. coli cells with UV-irradiated phage phi X174. Induction and decay kinetics of reactivation very much resembled that of mutagenesis of the UV-damaged phage. Repair as well as mutagenic activity increased for about 30 min. The maximal error-prone repair capacity, which was induced in the cell during the 30 min nalidixic acid treatment, rapidly died out during subsequent cell growth in absence of nalidixic acid. Induction of this repair mode was not observed in a recA- mutant. In the presence of nalidixic acid plus rifampicin both repair and mutagenic effects were abolished.     

Mutagenicity of benzotrichloride and related compounds.

Benzotrichloride (BTC), benzal chloride (BDC), benzyl chloride (BC) and benzoyl chloride (BOC) were surveyed for their mutagenicity in microbial systems such as rec-assay using Bacillus subtilis and reversion assays using E. coli WP2 and Ames Salmonella TA strains with or without metabolic activation in vitro. BTC and BDC required metabolic activation for their mutagenic activities in several strains of E. coli and Salmonella. The mutagenic metabolites of these compounds may not have been produced by hydrolysis. BC was weakly mutagenic without metabolic activation. Only BOC exhibited no mutagenic activity in the detection procedures used. The mutagenic metabolite of BTC might be very unstable under our experimental conditions. The strain E. coli WP2 try hcr was more sensitive than E. coli B/r WP2 try (hcr+) with regard to the mutagenicity of BTC.     

A new strain of Salmonella typhimurium reverted by mitomycin C and N-methyl-N'-nitro-N-nitrosoguanidine--a possible universal tester for mutagenic compounds

A strain of Salmonella typhimurium, SO1007, which carries the amber mutation trpD28 plus the plasmid pKM101 was reverted very efficiently by two mutagens with different mutagenic specificities and modes of action: mitomycin C (MC) and N-methyl-N'-nitro-N-nitrosoguanidine (NG). By selecting revertants on minimal agar supplemented with anthranilic acid (AA), two distinct phenotypic classes of TrpD28 revertants can be recovered: prototrophs (MM+) and anthranilate utilizers (AA+). Since each phenotypic class is known to be caused by a variety of mutational events, reversion of trpD28 on minimal-anthranilate medium may be useful for detecting mutagenic agents regardless of the types of mutations they may cause. Thus, strains like SO1007 may be useful as 'universal' detectors of mutagenic compounds. In the course of these experiments we also observed that pKM101 does not protect but, on the contrary, sensitizes the host bacteria slightly to the toxic effects of MC.     

Impact of an Urban Effluent on Antibiotic Resistance of Riverine Enterobacteriaceae and Aeromonas spp.

In order to evaluate the impact of an urban effluent on antibiotic resistance of freshwater bacterial populations, water samples were collected from the Arga river (Spain), upstream and downstream from the wastewater discharge of the city of Pamplona. Strains of Enterobacteriaceae (representative of the human and animal commensal flora) (110 isolates) and Aeromonas (typically waterborne bacteria) (118 isolates) were selected for antibiotic susceptibility testing. Most of the Aeromonas strains (72%) and many of the Enterobacteriaceae (20%) were resistant to nalidixic acid. Singly nalidixic acid-resistant strains were frequent regardless of the sampling site for Aeromonas, whereas they were more common upstream from the discharge for enterobacteria. The most common resistances to antibiotics other than quinolones were to tetracycline (24.3%) and beta-lactams (20.5%) for Enterobacteriaceae and to tetracycline (27.5%) and co-trimoxazole (26.6%) for Aeromonas. The rates of these antibiotic resistances increased downstream from the discharge at similar degrees for the two bacterial groups; it remained at high levels for enterobacteria but decreased along the 30-km study zone for Aeromonas. Genetic analysis of representative strains demonstrated that these resistances were mostly (enterobacteria) or exclusively (Aeromonas) chromosomally mediated. Moreover, a reference strain of Aeromonas caviae (CIP 7616) could not be transformed with conjugative R plasmids of enterobacteria. Thus, the urban effluent resulted in an increase of the rates of resistance to antibiotics other than quinolones in the riverine bacterial populations, despite limited genetic exchanges between enterobacteria and Aeromonas. Quinolone resistance probably was selected by heavy antibiotic discharges of unknown origin upstream from the urban effluent.     

Mutation induction with UV- and X-radiations in spores and vegetative cells of Bacillus subtilis.

Spores and vegetative cells of Bacillus subtilis strains with various defects in DNA-repair capacities (hcr-, ssp-, hcr-ssp-) were irradiated with UV radiation or X-rays. Induced mutation frequency was determined from the observed frequency of prototrophic reversion of a suppressible auxotrophic mutation. At equal physical dose, after either UV- or X-irradiation, spores were more resistant to mutations as well as to killing than were vegetative cells. However, quantitative comparison revealed that, at equally lethal doses, spores and vegetative cells were almost equally mutable by X-rays whereas spores were considerably less mutable by UV than were vegetative cells. Thus, as judged from their mutagenic efficiency relative to the lethality, X-ray-induced damage in the spore DNA and the vegetative DNA were equally mutagenic, while UV-induced DNA photoproducts in the spore were less mutagenic than those in vegetative cells. Post-treatment of UV-irradiated cells with caffeine decreased the survival and the induced mutation frequency for either spores or vegetative cells for all the strains. In X-irradiated spores, however, a similar suppressing effect of caffeine was observed only for mutability of a strain lacking DNA polymerase I activity.