Development of a toxicity test to be coupled to the Ames Salmonella assay and the method of constriction of the required strains

A 'toxicity' test protocol is described here to be used for determining the bactericidal effect of the chemicals which are tested for their mutagenic activity by the Ames method. Two sets of strains, isogenic with the Ames tester strains except for their his character, are constructed. One set is the his+ derivatives of the tester strains which are used for measuring the survival of the inoculum cells after exposure to the chemical. The other set is the stable his- derivatives of the tester strains which are used for simulating the background growth in the Ames mutagenicity plate test. The per cent survival of the his+ cells in the inoculum in the presence of the 'filler cells' is used as a measure of the toxic effect of the chemical.     

Tandem chromosomal duplications in Salmonella typhimurium: fusion of histidine genes to novel promoters.

Salmonella strains harboring tandem chromosomal duplications have been identified following selection for expression of a histidine biosynthetic gene whose promoter is deleted. In such strains, tandem duplications fuse the selected his gene to “foreign” regulatory elements, thereby allowing gene expression. Selection is made for hisD⁺ activity in deletion strain hisOG203. Among the revertants, strains harboring tandem chromosomal duplications have been identified by a number of their properties. (1) Their HisD⁺ phenotype is genetically unstable. (2) Such instability is dependent on recombination (recA) activity. (3) Genetic tests demonstrate that these strains are merodiploid for large regions (up to 25%) of the Salmonella genome. (4) Recipient strains that inherit the HisD⁺ phenotype of these duplication-carrying revertants also inherit the donor's merodiploid state. (5) In certain revertants the functional hisD⁺ gene and the sequence which promotes merodiploid transductant formation are linked to chromosomal markers located far from the normal his region.Previous reports have concluded that the instability of strains isolated by this selection is due to translocation of the hisD⁺ gene to an extrachromosomal element (the pi-histidine factor). We believe that in all strains we have tested (33 independent isolates) instability can better be accounted for as due to tandem duplication events which permit expression of hisD. At least two mechanisms are responsible for duplication formation. One mechanism is dependent on recombination function and generates identical revertants having a duplication of 16% of the chromosome. A second mechanism operates independently of recombination activity; individual duplications produced by this process have variable endpoints.     

Correlation between the toxicity of platinum drugs to L1210 leukaemia cells and their mutagenic properties.

There is a good correlation between the growth inhibitory properties of a series of Pt(II) derivatives on cultured L1210 mouse leukaemia cells and their efficiency of reversion to his⁺ of S. typhimurium TA100, or with their forward mutagenic effects on prophage λ. Reactivity towards DNA may thus explain the antitumor properties of these drugs.     

Rearrangement of the Bacterial Chromosome Using Tn10 as a Region of Homology

The transposable tetracycline resistance element, Tn10, can serve as a region of homology to promote rec-dependent deletion, duplication and directed transposition of bacterial genes. Tn10 insertions in regions of the chromosome near the histidine operon (his) have been isolated and characterized in Salmonella typhimurium. When strains are constructed containing two Tn10 insertions flanking the his operon in the same orientation (Tn10-his-Tn10), recombination can occur between Tn10 sequences resulting in the deletion of the intervening his region. The sites of the Tn10 insertions determine the endpoints of the deletion. In crosses designed to construct strains carrying Tn10-his-Tn10, another class of unstable recombinants arises in which the his region exists in tandem duplication, with a Tn10 insertion joining the duplicated copies (his-Tn10-his). The sites of the parental Tn10 insertions mark the endpoints of the duplication. When a strain carrying Tn10-his-Tn10 is used as a donor of his⁺ in conjugation or P22-mediated transduction, recombinants can arise in which the his region has been transposed to the site of any Tn10 insertion, far from the normal location of his in the recipient chromosome. In this manner, the his operon has been moved to the site of a pyrB::Tn10 insertion and has been placed on F'' plasmids. At these new locations, the his⁺ character shows the rec-dependent deletion of his⁺ expected for a Tn10-his-Tn10 duplication. These methods should be generally useful for the manipulation of bacterial genes.     

AICAR is not an endogenous mutagen in Escherichia coli

A number of observations in the Escherichia coli and Salmonella typhimurium literature could be explained by the hypothesis that a particular purine ribonucleotide precursor can be converted to the corresponding deoxyribonucleotide triphosphate, thereby becoming a base-analogue mutagen. The metabolite in question, AICAR (5-amino-4-carboxamide imidazole riboside 5′-phosphate), is also a by-product of histidine biosynthesis, and its (ribo)triphosphate derivative, ZTP, has been detected in E. coli. We constructed E. coli tester strains that had either a normal AICAR pool (pur ⁺ his ⁺ strains cultivated without purines or histidine) or no AICAR pool (purF hisG mutant strains, lacking the first enzyme of each pathway and cultivated in the presence of adenine and histidine). Using a set of lacZ mutations, each of which can revert to Lac⁺ only by a specific substitution mutation, we found that no base substitution event occurs at a higher frequency in the presence of an AICAR pool. We conclude that the normal AICAR pool in E. coli is not a significant source of spontaneous base substitution mutagenesis.     

The mutagenicity and DNA-damaging activity of cyclic aliphatic sulfuric acid esters.

The cyclic aliphatic sulfuric acid esters 1,2-ethylene sulfate (ESF), 1,3-propylene sulfate (PSF) and 1,3-butylene sulfate (BSF) have been tested for their mutagenic and DNA-damaging activity. Mutagenicity of the compounds was established with his-auxotrophic indicator strains of Salmonella typhimurium using the in vitro plate test and the host-mediated assay technique with mice as host animals. The DNA-damaging activity was tested in a repair test with Proteus mirabilis mutants defective in DNA repair.In the repair test with a set of P. mirabilis strains (PG713 hcr^?rec^?: PG273 hcr⁺rec⁺) PSF and BSF showed a preferential growth inhibition of the repair-defective strain suggesting DNA-damaging activity of these chemicals. No such activity was found for ESF using the same concentrations of 5 and 15 μmol/plate.All cyclic sulfates revert the tester strain TA1535 of S. typhimurium in vitro indicating their ability to induce base substitutions. Compared with the reference compounds dimethyl sulfate (DMS), diethyl sulfate (DES), 1,3-propane sulfone (PPS) and 1,4-butane sulfone (BTS) the mutagenic activity in the plate test can be described as follows: PPS > PSF > BSF > BTS > ESF > DES > DMS.Dose-response studies in the host-mediated assay with tester strain TA1950 of S. typhimurium as genetic indicator system revealed a linear dosedependency of mutagenic activity. For PPS and PSF the lowest effective dose (LED) has been established as 10 μmol/kg. The LED for BSF and BTS was 50 μmol/kg, DMS and DES were mutagenic in doses of 2500 μmol/kg, while ESF was only weakly mutagenic with a LED of 5000 μmol/kg.The dose-response studies in the host-mediated assay and the results obtained in the in vitro spot test demonstrate similarities in the mutagenic action of the cyclic sulfates PSF and BSF and the respective sulfones, while the stronger alkylating compound ESF was a weak mutagen both in vitro and in vivo.     

Isolation and prevalidation of an Escherichia coli tester strain for the use in mechanistic and metabolic studies of genotoxins

We have isolated an Escherichia coli tester strain for the use in mechanistic and metabolic studies of genotoxins. We started with one of the more used and better characterized E. coli K-12 laboratory strains, AB1157. We isolated a lipopolysaccharide defective mutant of strain AB1886 which is an excision repair deficient derivative of AB1157 and introduced a newly constructed plasmid pKR11, encoding mucAB, resulting in strain MR2101/pKR11. A genotoxicity assay was designed, monitoring the reversion to arginine prototrophy and a preliminary validation was carried out against Ames tester strain TA100 with a set of diagnostic compounds. The results seem to indicate that strain MR2101/pKR11 is an adequate tester strain which can be a useful tool in mechanistic studies. Moreover, this strain can serve as mother strain to isolate improved and more especialized tester strains.     

Mutagenicity of oil-shale ash

3 oil-shale ash samples were extracted with solvents and analyzed for mutagenicity with a number of test systems. In Salmonella typhimurium, the ash extracts were highly mutagenic with the Ames his reversion and the ara-resistant systems. Mutation induction by the ash in Salmonella was independent of metabolic activation and was of the frameshift type. These ash extracts showed a substantial killing effect, but failed to induce ad-3 reversion in Neurospora crassa, gene conversion and mitotic crossing-over in Saccharomyces cerevisiae and TG^r mutation in cultured CHO cells.     

Mutagenicity of styrene and styrene oxide

Incubation of S. typhimurium strain TA 1535 with styrene increased the number of his⁺ revertants/plate in presence of a fortified S9 rat-liver fraction. Styrene was also highly cytotoxic for Salmonella cells. Styrene oxide, the presumed first metabolite, had a mutagenic effect towards strains TA 1535 and TA 100 both with and without metabolic activation. Styrene is probably mutagenic because it is metabolized to styrene oxide.     

Deletion mutants of nitrogen fixation in Klebsiella pneumoniae: Mapping of a cluster of nif genes essential for nitrogenase activity

Deletions of the nitrogen fixation (nif) region of the Klebsiella genome were isolated by selecting for resistance to virulent phages whose resistance loci are adjacent to nif. The extent of the various deletions was monitored by assaying several different enzymes or gene products coded for by this segment of DNA. Three classes of deletion mutants were detected: (1) gluconate-6-phosphate dehydrogenase minus (gnd^?), histidine minus but histidinol dehydrogenase plus (his^?, his D⁺), nitrogenase plus (nif⁺), shikimate utilization plus (shu⁺); (2) gnd^?, his D^?, nif^?, shu⁺; (3) gnd^?, his D^?, nif^?, shu^?. From these studies we conclude that the cluster of nif genes essential for nitrogenase activity is located on the genetic linkage map of Klebsiella between his and shu; the gene order in this region in thus phage-resistance locus (rfb?), gnd, his operon, nif, shu. Genetic analysis substantiates the finding that the nif cluster is located proximally to the operator end of the his operon.     

Genetic analysis of the histidine operon control region of Salmonella typhimurium

Mutants of the histidine operon control region (hisO) include two classes: (1) those completely unable to express the operon (His auxotrophs), and (2) prototrophs that are unable to achieve fully induced levels of operon expression (still His⁺ but sensitive to the drug amino-triazole). Using new, as well as previously existing hisO mutants, we constructed a fine-structure deletion map of hisO. Mutations that presumably alter the his promoter map at one end of hisO; mutations that alter the his attenuator map at the other end of hisO. Between the promoter and the attenuator lie a number of mutations that affect either the translation of the his leader peptide gene, or the formation and stability of his leader messenger RNA structures. All of the point mutations mapping in this central region revert to His⁺ at a very high frequency (10^?5 to 10^?6); this frequency is increased by both base substitution and frameshift-inducing mutagens. Many of the His^? mutants are suppressed by informational suppressors; all three types of nonsense mutations have been identified, demonstrating that translation of a region of hisO between the promoter and attenuator is essential for his operon expression. All of the hisO mutations tested are cis-dominant.     

Effects of newly synthesised platinum(ii) complexes on mitochondrial functions

Protease deficient recA431 mutants of Escherichia coli are defective in their capacity for induction of SOS responses and were intermediate in their sensitivities to ultraviolet light (UV) and cis-platinum(II)diamminodichloride (cis-PDD). Survival after treatment determined as colony forming ability was greater in rec⁺ strains and decreased in recA13 mutants which are defective in both recA proteolytic and recombination capabilites. In contrast, recA431 mutants were as sensitive to N-methyl-N′-nitro-N-nitrosoguanidine (NTG) as the recA13 cells. When cells carried either the pKM101 or N3 plasmid, survival after treatment with the three mutagens was increased. Presence of these plasmids in cells also resulted in hypermutagenicity as indicated by reversion of the argE3 mutation using a modified Ames test. Mutagenesis by NTG and cis-PDD was increased, as was survival of cells treated with UV light, cis-PDD and NTG in both recA⁺ and recA431 (protease deficient) strains. No plasmid mediated enhancement of mutagenesis or cell survival was observed in recA13 mutants. Thus, the ability of the plasmids to enhance cell survival and mutagenesis was dependent on recombination proficiency of the recA gene product and not its regulatory proteolytic activity. Unlike UV or NTG, presence of one of these plasmids was needed to detect reversion of the argE3 mutation by cis-PDD.     

Donor Strains of the Soft-Rot Bacterium Erwinia chrysanthemi and Conjugational Transfer of the Pectolytic Capacity

Donor strains of Erwinia chrysanthemi ICPB EC16, a member of the soft-rot (pectolytic) section of the enterobacterial genus Erwinia, were obtained by chromosomal integration of an F′lac⁺ plasmid originating from Escherichia coli. These stable donor strains, selected from an unstable F′lac⁺ heterogenote by repeated platings of single Lac⁺ colonies on lactose minimal agar, do not segregate (as does the parent F′lac⁺ heterogenote) into Lac⁻ or F⁻ clones, in either the presence or absence of acridine orange. One representative donor strain (from the 12 that have been selected) has been examined in more detail; it can transfer ade⁺, gal⁺, gtu⁺ (utilization of galacturonate), his⁺, lac⁺, leu⁺, lys⁺, mcu⁺ (multiple carbohydrate utilization), pat⁺ (production of polygalacturonic acid trans-eliminase), thr⁺, and trp⁺ in a polarized manner to appropriate recipient strains of E. chrysanthemi; the frequencies of ade⁺, leu⁺, and thr⁺ transfer were higher than those of the other markers tested to date. This donor strain transfers lac⁺ genes during a 6-h mating on membranes; most of the Lac⁺ recombinants are donors of chromosomal markers. The kinetics of entry as well as the frequencies of transfer of chromosomal markers indicate that thr⁺ and leu⁺ enter the recipient as proximal markers and that lac⁺ enters as a distal marker. Analysis of the recombinants demonstrates close linkage between thr and leu, ade and thr, his and pat, and his and trp loci. The results suggest that the integration of F′lac⁺ into the chromosome of E. chrysanthemi has occurred at a region adjacent to the leu-thr loci, and that the chromosome is transferred in the following sequence: origin----leu--thr--ade--lys--mcu--pat--his--trp--gal--gtu--lac--F. Plant-tissue maceration occurs in Pat⁺ recombinants and not in Pat⁻ recombinants, even though both form another pectolytic enzyme, hydrolytic polygalacturonase. This genetic evidence supports the idea that the E. chrysanthemi polygalacturonic acid trans-eliminase plays an essential role in bringing about plant-tissue maceration.     

The pleiotropic effects of his overexpression in Salmonella typhimurium do not involve AICAR-induced mutagenesis

Inhibition of cell division associated with overexpression of hisH and hisF in Salmonella typhimurium is strongly reminiscent of a cellular response to DNA damage. On these grounds, we investigated the involvement of a metabolite which appeared to represent a possible candidate for an endogenous mutagen: the base analog 5-amino-4-carboxamide imidazole riboside 5′-phosphate (AICAR), a by-product of HisH and HisF activity. However, we showed that AICAR is not an endogenous mutagen in S. typhimurium. Other types of DNA damage induced by his overexpression seem also unlikely, since similar mutation rates are found in hisO ⁺ and hisO ^c strains. We also show that AICAR production is not involved in the pleiotropic effects of his overexpression, since these are still observed in strains devoid of AICAR. Thus inhibition of cell division resulting from HisH and HisF overexpression must operate through a mechanism unrelated to the role of these proteins in histidine biosynthesis.     

Residual recombination in Neurospora crassa spo11 deletion homozygotes occurs during meiosis

Spo11 is considered responsible for initiation of meiotic recombination in higher organisms, but previous analysis using spo11 ^RIP mutants suggests that the his-3 region of Neurospora crassa experiences spo11-independent recombination. However, despite possessing several stop codons, it is conceivable that the mutants are not completely null. Also, since lack of spo11 interferes with chromosomal pairing and proper segregation at Meiosis I, spores can be partially diploid for a period after meiosis. Thus, it is possible that the recombination observed could be an abnormal event, occurring during the period of aneuploidy rather than during meiosis. To test the former hypothesis, we generated spo11 deletion homozygotes. Using crosses heteroallelic for his-3 mutations, we showed that His⁺ progeny are generated in spo11 deletion homozygotes at a frequency at least as high as in wild type and, as in the spo11 ^RIP mutants, local crossing over is not reduced. To test the latter hypothesis, we utilised mutations in either end of a histone H1-GFP fusion gene, inserted between the recombination hotspot cog and his-3, in which GFP⁺ spores arise as a result of recombination in a cross between the two GFP alleles. In a control cross homozygous for spo11 ⁺, the frequency at which GFP⁺ spores arise is comparable to the frequency of His⁺ spores and glowing nuclei first appear during prophase, prior to metaphase I, as expected for a product of meiotic recombination. Similarly in spo11 deletion homozygotes, GFP⁺ spores arise at high frequency and glowing nuclei are first seen before metaphase, indicating that allelic recombination occurs during meiosis in the absence of spo11. We have therefore shown that spo11 is not essential for either his-3 allelic recombination or crossing over in the vicinity of his-3, and that spo11-independent allelic recombination is meiotic, indicating that there is a spo11-independent mechanism for initiation of recombination in Neurospora.     

Renin inhibitors as an example of presumptive irrelevant positive findings in the Salmonella/mammalian microsome assay (Ames test)

An increase in the number of mutant colonies in the Ames test is generally taken as a strong indication for a genotoxic (e.g., DNA damaging) property of the test compound or its metabolites. However, a few examples are known in which mechanisms usually related to some sort of growth enhancement will lead to increases in mutant frequencies of spontaneous origin.The renin inhibitor Ro 42-5892 increased the number of mutant colonies of strain TA1538 and to a lesser degree of TA98 in the standard plate incorporation assay (Ames test). Since there is no chemical basis for a ‘DNA reactivity’ of this compound, experiments were performed to obtain information about possible indirect mechanisms of enhancing the number of spontaneous mutant colonies.Circumstantial evidence is presented to attribute the weak activity not to an inherent genotoxic property but rather to an as yet undefined indirect effect on the expression of spontaneous mutants.Since Ro 42-5892 contains a histidine residue it was a reasonable assumption to suspect a growth enhancing property of the test compound. However, none of the strains showed an elevation of the number of revertant colonies or an increase in the density of the background growth. In addition, structurally related non-histidine containing renin inhibitors showed absolutely no increase in the number of revertant colonies. Furthermore, no growth induction (either in liquid or under selective conditions) and no histidine cleave off by a TA1538/TA98 specific metabolism could be shown.A second line of evidence showing parallelism to growth enhancing compounds concerns the comutagenicity of histidine containing renin inhibitors. When Ro 42-5892 was tested in combination with established mutagens, a multiplicative synergism was found. This effect was observed not only in strains TA1538 and TA98 but also in the standard Salmonella tester strains where the spontaneous mutant frequency was not increased by Ro 42-5892. Analogous effects were previously shown for free histidine, isohistidine, phenobarbital and tetracycline and in part explained by molecular mechanisms.     

Accumulation of single-stranded regions in DNA and the block to replication in a human cell line alkylated with methyl methane sulfonate

Once a permeability barrier is overcome, proflavin is highly mutagenic for Salmonella typhimurium and Escherichia coli. Mutagenesis of the his operon (in Salmonella) and lac operon (in E. coli) depends on derepression of the respective operons. No proflavin mutagenesis was detected in rec^? strains. Over 100 proflavin-induced his mutants have been classified: 50% are base substitution types, 30% are stable (10% demonstrably multisite), and only 20% are probably frameshift mutations. None of the proflavin-induced frameshift mutations is of the type previously shown to be suppressed by frameshift suppressor mutations.     

AICAR is not an endogenous mutagen in Escherichia coli

A number of observations in the Escherichia coli and Salmonella typhimurium literature could be explained by the hypothesis that a particular purine ribonucleotide precursor can be converted to the corresponding deoxyribonucleotide triphosphate, thereby becoming a base-analogue mutagen. The metabolite in question, AICAR (5-amino-4-carboxamide imidazole riboside 5-phosphate), is also a by-product of histidine biosynthesis, and its (ribo)triphosphate derivative, ZTP, has been detected in E. coli. We constructed E. coli tester strains that had either a normal AICAR pool (pur ⁺ his ⁺ strains cultivated without purines or histidine) or no AICAR pool (purF hisG mutant strains, lacking the first enzyme of each pathway and cultivated in the presence of adenine and histidine). Using a set of lacZ mutations, each of which can revert to Lac⁺ only by a specific substitution mutation, we found that no base substitution event occurs at a higher frequency in the presence of an AICAR pool. We conclude that the normal AICAR pool in E. coli is not a significant source of spontaneous base substitution mutagenesis.