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Different conditions of mutagenesis have been compared in order to optimize the use of the L-arabinose resistance test with Salmonella typhimurium. The mutagenesis protocols compared were the plate-incorporation, the pre-incubation and the liquid tests. Fourteen chemicals were used in the comparison: six direct-acting mutagens and eight pre-mutagens. Five concentrations of S9 (3, 7.5, 10, 15 and 33% v/v) were compared in the liquid test with pre-mutagens, and three densities of bacteria (ranging from 10(8) to 10(6) cells) were used in the comparison between the plate-incorporation and the pre-incubation mutagenesis test. In general, the liquid test proved the most sensitive mutagenesis protocol. When carrying out this test in a mass screening of mutagens, we propose to select the L-arabinose-resistant mutants in plates supplemented with 0.5 mg of D-glucose, and to express the mutagenic response as the absolute number of induced mutants. The plate-incorporation and the pre-incubation mutagenesis protocols could be considered as alternative procedures in the case of previous negative results with the liquid test. Two recommendations can be finally made in order to avoid false negative results: (a) a large population of bacteria (ranging from 10(8) to 10(7) cells) must be exposed to the mutagens in both the plate-incorporation and the pre-incubation mutagenesis tests, and (b) ideally, several S9 concentrations (ranging from 3 to 30% v/v) would be employed for testing in liquid samples of unknown mutagenicity.

The present study was designed to evaluate the capacity of the L-arabinose resistance test of Salmonella typhimurium in the detection of frameshift-type mutagens. To this end the response of the Ara test was examined with respect to 15 chemicals which had been previously described as able to revert the Ames tester strain TA97. The mutagenicity of each compound was determined by the liquid test under experimental conditions which optimize the mutagenic response of the Ara test with the tester strain BA9. Strain TA97 was used simultaneously with BA9. The Ara forward-mutation assay efficiently detected the mutagenic activity of 14 out of the 15 chemicals assayed. PR toxin was the only compound which gave a weak dose response without doubling the spontaneous mutant level. In comparison with the Ara test, a total of 3 chemicals (HZ, PE and PR toxin) were not found to be mutagenic with strain TA97. In most cases (11/15) the mutagenic response of the Ara test was comparatively greater than that of strain TA97. Three chemicals (DEO, PRF and 9-AA) were detected with quite similar degrees of sensitivity by both mutation assays. ICR-191, which seems highly specific in reverting frameshift mutations with added cytosines in a run of cytosines, was the only chemical with a lower mutagenic activity in the Ara test than in strain TA97. The results enhance the interest of the L-arabinose forward-mutation assay as an alternative to the set of specific tester strains used by the histidine reverse-mutation assay in massive, general and primary screening for genotoxic agents.

Simultaneous expression of seven genes in Escherichia coli was measured by a reverse transcription-multiplex PCR fluorescence procedure. Genes studied were (i) oxyR (transcriptional regulator); (ii) katG , dps , gorA , and ahpCF (controlled by OxyR); (iii) sodA (controlled by SoxRS); and (iv) trxA (not related to OxyR or SoxRS). Except for trxA , transcription of all genes was activated during the course of growth of wild-type bacteria, though notable variations were observed with respect to both the time and extent of activation. Whereas oxyR , katG , dps , and gorA were activated during exponential growth, ahpCF and sodA were stimulated in stationary phase. Maximal induction ranged from 4.6- to 86.5-fold, for gorA and dps , respectively. Treatment with H 2 O 2 stimulated expression of the genes ( katG , dps , ahpCF , and gorA ) previously identified as members of the OxyR regulon, except for oxyR itself. Induction by H 2 O 2 was a remarkably rapid and reversible process that took place in an OxyR-dependent and ς S -independent manner. NaCl induced expression of the genes controlled by OxyR, including the oxyR locus. This transcriptional up-regulation was preserved in a strain with the Δ oxyR :: kan mutation, but it was abolished ( ahpCF ) or significantly reduced ( oxyR and dps ) in a strain with the rpoS ::Tn 10 mutation, potentially reflecting positive transcriptional regulation of the oxyR regulon by ς S . Expression of trxA was not increased either by H 2 O 2 stress or by a shift to high-osmolarity conditions.

Three species of marine bivalve molluscs (Chamelea gallina, Ruditapes decussatus, and Crassostrea gigas) have been studied in order to evaluate the levels of pollution on the South Atlantic Spanish littoral. Several transition metals (Cu, As, Cd, Sn, Hg, Pb) were determined as a general index of total contamination. Animals from putative contaminated areas exhibited higher metal contents than those from cleaner waters. C. gigas showed 5-20-fold higher total metal content than the other two species. The mutagenicity of ethanolic extracts was assayed by using both the His reversion and the Ara forward mutation tests. Mollusc tissues from the three species did not contain genotoxins active on TA98 (frameshift mutations) or TA100 (mainly G:C base-pair substitutions), but did contain direct-acting genotoxins of a polar nature and oxidative type. This was based on the following observations: 1) mammalian metabolic activation was not required for mutagenicity, 2) mutagens were eluted with the polar fraction from XAD-2 columns, and 3) mutagenic responses were observed with Salmonella typhimurium TA102 (A:T base-pair substitutions; sensitive to oxidative damages) and Escherichia coli catalase-deficient (AraR forward mutations) strains. No relevant differences were found in the mutagenicity of mollusc extracts from areas with different pollution levels. Otherwise, our data suggest that, in general, animals living in contaminated environments had fewer genotoxins of oxidative type than those from less polluted areas. Such a result might be explained by the observation of increased levels of a number of detoxifying and antioxidant enzymes, such as glutathione-S-transferase, glutathione-peroxidase, catalase, and superoxide dismutase. Thus, contaminated animals seem to be better protected against the oxidative damages induced by metals, in agreement with their lower malondialdehyde levels. To what extent the responsible mutagenic compounds are of endogenous origins, or "Nature's pesticides" (the major toxic chemicals ingested by phytoplankton filter-feeders), and/or the result of human activities remains to be determined.

Forward mutations induced by ethylmethane sulfonate (EMS) in the LacI gene of Escherichia coli were recovered from bacteria proficient or deficient in the alkyltransferase encoded by the constitutive ogt gene. EMS doses of 100 or 200 mM (Ogt + ) and of 50 mM (Ogt - ) were selected from the corresponding dose-response curves for DNA sequence analysis. A total of 239 induced mutations affecting the N-terminal region of the lacI gene were characterized. All mutations were G:C→A:T transitions, consistent with the predominant role of the O 6 -ethylguanine miscoding lesion in mutagenesis by EMS. In the Ogt + spectrum at the lowest tested dose of 100 mM EMS, guanines preceded by an A or T base at the 5' side were on average 3.2 times more likely to mutate than those preceded by a G or C base. This bias diminished at the higher EMS dose (200 mM) and disappeared in the Ogt - genetic background. Previously reported data for Ogt + bacteria in a Uvr-proficient background show an opposite bias in favor of mutations at guanines preceded by a G or C base. The overall 5' flanking base influence was estimated as 8-fold. These data suggest that DNA repair by Ogt alkyltransferase plays an important role in the processing of ethylation-induced lesions responsible for GC→AT transitions, influencing their ultimate distribution with respect to sequence context. The data further suggest that Ogt and UvrABC excision repair, the two major mechanisms of protection against the biological consequences of long-chain alkylating agents, show different DNA sequence specificity and that the relative importance of these two systems is highly dependent upon the chemical dose

Sixteen halogenated aliphatic hydrocarbons were assayed for genotoxicity using the Ara mutagenicity assay with Salmonella typhimurium. Seven substances (1,2-dibromo-3-chloropropane, 1,2-dibromoethane, 1,2-dichloroethane, vinyl bromide, hexachloro-1,3-butadiene, iodoform and vinilydene chloride) were mutagenic at non-lethal doses. Comparatively, nine compounds (chloroform, carbon tetrachloride, 1,1,1-trichloroethane, 1,1,2-trichloroethane, tetrachloroethylene, trichloroethylene, 1,1,1,2-tetrachloroethane, 1,1,2,2-tetrachloroethane and hexachloroethane) were non-mutagenic after being assayed both in the presence and absence of metabolic activation with a rat liver microsomal fraction (S9). All negative compounds (except hexachloroethane) gave a lethal response, which could be an indication that bacteria were adequately exposed. The concordance between mutagenicity in the Ara test and carcinogenicity in rodents for this group of halogenated hydrocarbons was (31%) significantly lower than the concordance (72%) previously found in the Ara test with respect to a wider range of chemical classes. This result is in agreement with data reported for other genotoxicity assays. The presence of non-genotoxic carcinogens versus genotoxic non-carcinogens is discussed as a possible explanation. Five positive compounds (1,2-dibromo-3-chloropropane, 1,2-dibromoethane, 1,2-dichloroethane, vinyl bromide and hexachloro-1,3-butadiene) were analyzed for a quantitative relationship between carcinogenic potency in rats and the potency of response in the Ara mutagenicity test. This was possible because the Ara test, for volatile compounds (such as vinyl bromide), did not require the use of special vaporization techniques, which are difficult to evaluate quantitatively for mutagenic activity. A highly significant correlation was found between the mutagenic efficiencies of the five compounds in the Ara test and their carcinogenic potencies in rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The supF gene of Escherichia coli has been widely used as a mutagenic target in several shuttle-vector plasmids. Mutations in this gene are usually screened by a colony colour assay based on the suppression of a lacZ amber mutation in an appropriate bacterial indicator strain. This screening method cannot measure the low mutation frequencies usually detected in prokaryotes, and therefore precludes the use of supF gene for studying mutational spectra in bacteria. In this paper we report the development of a simple method for the selection of supF forward mutations in shuttle-vector plasmids. The method has implied the construction of an araD- araC(Am) mutant strain (MBL50) of E.coli. The L-arabinose sensitivity caused by the accumulation of a toxic intermediate in araD- mutants is abolished in MBL50 because the araC(Am) mutation blocks the L-arabinose catabolic pathway. Strain MBL50 becomes sensitive to L-arabinose when transformed with a supF+ plasmid but remains resistant upon transformation with a supF- mutant. This new L-arabinose resistance selection method was able to detect supF- mutant fractions up to three orders of magnitude below those determined with the colony colour screening assay. The method was further validated by carrying out in vivo mutagenesis experiments with N-methyl-N-nitrosourea (MNU) and a shuttle-vector-bearing strain (UC2109) completely defective in O6-methylguanine (O6meG) alkyltransferase repair capacity. The DNA sequence alterations of 22 independent supF- mutants induced by MNU were determined. All mutations were G:C-->A:T transitions in agreement with the predicted significance of the mispairing potential of the O6meG lesion. A preference for the sequence 5'-GG-3' was detected, revealing a 5'-flanking base influence. The accumulation of all 22 MNU-induced mutations in three sites of the supF genes might be related to the lack of O6meG alkyltransferase repair capacity of strain UC2109. The L-arabinose resistance method described in this paper allows rapid scoring and sequencing of forward mutations in the supF gene on shuttle-vectors, thus permitting its use as a genetic target for repair and mutagenesis studies in bacteria. Since shuttle-vectors replicate both in bacteria and mammalian cells, this method makes it possible to compare prokaryotic and eukaryotic mutational spectra.

This work investigates whether a mutator phenotype is associated to the simultaneous deficiency in thioredoxin and glutaredoxin, the two known hydrogen donors of ribonucleotide reductase. To this end, new Escherichia coli K-12 strains carrying delta trxA and/or grx::kan null mutations were constructed to monitor mutagenesis by selecting forward mutations to L-arabinose resistance. Highly sensitive and specific enzyme-linked immunoassays were developed to confirm that trx-grx- cells lacked thioredoxin and glutaredoxin. A number of remarkable properties were observed in the newly constructed thioredoxin- and glutaredoxin-deficient bacteria compared with the wild type cells. Thus, they (i) grew on minimal medium plates, suggesting that the presence of thioredoxin and glutaredoxin may not be absolutely essential for sulfate reduction; (ii) showed normal mutagenic sensitivities toward a wide variety of DNA-damaging agents, as compared with wild type cells and trx- or grx- single mutants; (iii) displayed 14% of GSH-dependent and 30% of NADPH-dependent ribonucleotide reduction capacity with CDP as substrate in the presence or the absence of exogenous ribonucleotide reductase, respectively; and (iv) showed very high levels of ribonucleotide reductase activity, which was increased from 19- to 23-fold. The existence of a new glutathione-dependent hydrogen donor for ribonucleotide reductase and the high activity levels of this enzyme in trx-grx- defective cells could explain that thioredoxin and the first discovered glutaredoxin are not essential for deoxyribonucleotide synthesis, even under mutagenic stress.

The standard Ames tester strains of Salmonella typhimurium are separated by many steps in their pedigree, some involving mutagen treatments, and contain independently isolated uvrB-bio-gal deletions and rfa mutations. In this work the araD531 mutation was introduced into the Ames tester strains TA100 and TA98. The responsiveness of the resulting strains (BA15 and BA14) to a number of chemical mutagens was then assessed by monitoring the induction of forward mutations to L-arabinose resistance (Ara test). Here we have shown that these two strains of the Ames test differ greatly in their responses to mutagens, in ways that are not associated with the mutagenic specificities of the original his mutations. In general, the genetic background of strain TA100 appears to be more sensitive to the killing effects of chemicals than that of TA98. The greatest differences were found with nifurtimox (NFX) and its analogue, compound 1K. The Ara test responded to the mutagenic effects of these two nitrofurans when carried out in the genetic background of strain TA98 but not in that of TA100. A higher sensitivity to the lethal effects of NFX and 1K together with the greater nitro-reduction capability of strain TA100 as compared with TA98 might explain the differences. In conclusion, our results indicate that the standard Ames S. typhimurium tester strains are not isogenic and that genetic differences at loci other than his might be significant for mutagenicity testing.(ABSTRACT TRUNCATED AT 250 WORDS)

The aim of the present study was to investigate whether bacterial mutagenicity assays can be applied in sediment quality assessment. The arabinose-sensitive forward mutation strain Salmonella typhimurium BA 9 was added to freshly deposited sediments of the River Elbe, collected during March, April, and May 1994. Up to twelve locations were sampled each month and mutagenicity was determined employing the solid phase and sequentially prepared toluene- and methanol-extracts of the sediments. Mutagenicity was detected at all sites within the solid sediments without addition of S9-mix; however, the addition of mammalian enzymes (S9) enhanced the mutagenic effect. In contrast, mutagenicity of toluene extracts containing the lipophilic fraction of the sediment samples was higher in the absence of S9-mix; peaks of mutagenic activity in these samples were observed at Dessau (mouth of the River Mulde) and close to the city of Hamburg (Bunthaus). Similar results were obtained with methanolic extracts of the sediments, although the effects were usually lower in comparison to the corresponding toluene extracts. These results show that the mutagenicity assays are capable of assessing water/sediment contamination and reveal that the mutagenicity detected in sediments reflects local industrial activities as well as hydrologic conditions.

The mutagenic and lethal effects of nine halogenated methanes (CCl4, carbon tetrachloride; CHCl3, chloroform; CH2Cl2, dichloromethane; CBr4, carbon tetrachloride; CHBr3, bromoform; CH2Br2, dibromomethane; CI4, carbon tetraiodide; CHI3, iodoform; and CH2I2, diiodomethane) have been investigated with the Ara forward-mutation assay of Salmonella typhimurium. Five substances (CH2Cl2, CBr4, CH2Br2, CHI3 and CH2I2) gave clear mutagenic responses. In all these cases the mutagenicity diminished in the presence of mammalian metabolic activation (S9 mixture). Two halomethanes (CCl4 and CHBr3) were classified as 'questionable' mutagens since they did not double the spontaneous value although a dose-response curve was obtained. All halomethanes tested exerted a lethal effect. A high concordance was found between lethality and chemical reactivity, as expected from the type and number of halogenated substituents. Compounds with equal numbers of substituents were lethal in the order I > Br > Cl. Additionally, the lethality of compounds with identical halogen substituents increased by successive halogenations. No such concordances were observed with respect to mutagenic activity. Structure-activity quantitative relationships were investigated by using the previously reported values of the polarographic half-wave reduction potential (-E1/2), a physicochemical parameter related to the energy required to put an electron into the lowest unoccupied molecular orbital. The lethality, quantified as LD37, correlated highly with the -E1/2 values for the nine halogenated compounds (r = -0.955 P < 0.001). These data suggest that halomethanes which are reduced easily will induce higher lethality than those with low reduction potential, in agreement with the predicted effects on lipid peroxidation and hepatotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

The dithiol forms of thioredoxin and glutaredoxin are hydrogen donors for ribonucleotide reductase. We have determined the intracellular levels of ribonucleotide reductase (RRase), thioredoxin (Trx), glutaredoxin 1 (Grx1), and glutathione (GSH) and the glutathione redox status in new Escherichia coli K12 strains lacking thioredoxin (trxA-), glutaredoxin 1 (grxA-), and/or GSH (gshA-) or overproducing Trx or Grx1 from multicopy plasmids. We propose a regulatory network in which RRase levels are balanced with those of Trx, Grx1, and GSH so that deficiency or overproduction of one component would promote the opposite effect on the others to maintain a balanced supply of deoxyribonucleotides. GSH deficiency strongly increased both Grx1 levels and RRase activity, even more than Trx deficiency. Double gshA-trxA- bacteria were viable, whereas additional deficiency in lipoate synthesis (gshA-trxA-lipA-) caused the inability to grow in minimal medium plates supplemented with acetate plus succinate instead of lipoic acid. Thus, lipoate might be the only substitute of GSH for glutaredoxin reduction in gshA-trxA- cells, although the extremely high Grx1 content (55-fold) of these bacteria suggests that electron transfer from lipoate might be an inefficient reduction mechanism of glutaredoxins. Moreover, the enhanced Grx1 level of gshA-trxA- cells could obviate the need for a large increase in RRase activity, in contrast to grxA-trxA- double mutant cells. Impairment of the sulfate assimilation pathway, leading to very low GSH concentrations, and an oxidized glutathione redox state might explain the inability of grxA-trxA- cells to grow in minimal medium. Restoration of nearly normal levels of both GSH content and redox status cure the growth defect.

The forward mutation assay to L-arabinose resistance (Ara test) in Salmonella typhimurium was used to demonstrate that evaporated residues of brandy had direct-acting mutagenic activity. The mutagenicity covered a 100-fold range, from 13482 to 127 AraR induced mutants/ml brandy equivalent. Rat liver S9 mix suppressed the mutagenic activity of brandy in the Ara test. The inactivating capacity was independent of microsomal monoxygenase enzymes and appeared to be mediated through a heat stable component of the S9 fraction. Catalase was identified as the putative S9 component responsible for its inactivating capacity. The implication of reactive oxygen species in the direct-acting mutagenicity of brandy was supported by the higher sensitivity of Escherichia coli bacterial strains deficient in two major cellular antioxidant defense (glutathione and/or catalase) compared to their parental wild-type. Phenolic compounds of a polar nature could be responsible for the mutagenicity through the production of reactive oxygen intermediates. Non-matured beverages (gin and non-matured rum) were non-mutagenic. It is conceivable that mutagenic phenolics might be extracted from the wood during maturation in the barrel. Autoxidation of phenolic compounds could be a common mechanism in the mutagenicity of complex mixtures of plant origin.

The forward-mutation assay using the L-arabinose-sensitive strain SV3 of Salmonella typhimurium has been calibrated against a selected set of mutagens. Strain SV3 is sensitive to chemicals causing base-pair substitutions, frameshift mutations and deletions. New strains deficient for the excision-repair system or the lipopolysaccharide barrier or both have been selected from strain SV3. The additional mutations do not affect the independence of the assay from experimental artifacts due to physiological or lethal damage or differences in plating density. The new strains are more sensitive than SV3 to certain mutagens. Techniques for using this set of strains are presented and their relative advantages discussed.

Thirty-two commercially produced white, rose, and red wines from Spain were assayed for genotoxicity. The Ara forward mutagenicity assay with Salmonella typhimurium served as the test system. All the wines were mutagenic in the absence of mammalian microsomal activation (S9 mix) and/or glycosidase activities with the exception of one rose wine which gave a clear dose-response relationship, although its mutagenic potency was considered statistically nonsignificant. The mutagenic activity covered nearly a 30-fold range. Compared to white and rose wines, red wines showed the highest levels of mutagenicity; this wine type included four "very potent" (greater than 3,000 AraR mutants/ml) mutagenic wines. The level of wine mutagenicity did not correlate with either the region or the year of production (vintage). Individual winery methods are suggested as primarily responsible for variations in mutagenic activity. The present study with the Ara test supports the possibility that wine components other than the flavonols quercetin and rutin are the major putative mutagens: (1) white wines, as well as rose or red wines, were detected as being mutagenic; (2) in no case was activation required for the detection of mutagenicity; (3) mutagen(s) were detected mainly (red wine) when not exclusively (white and rose wine) in the polar fraction from XAD-2 chromatography. The high sensitivity of the Ara test has allowed the screening of the mutagenicity of a variety of wines with no previous process of extraction or concentration. The comparison of the mutagenic activity of the entire complex mixture to that of its lyophilized residue has revealed a positive synergistic role for ethanol in the mutagenicity of certain wines. Finally, this work suggests that the Ara test is a useful tool for mutagenicity screening in wines. Thus, this test might play an important role in elucidating the genotoxic mechanism of action of alcoholic beverages, and for studying optional production methods to decrease the mutagenicity of commercial wines.

The forward mutation assay to L-arabinose resistance (Ara test) in Salmonella typhimurium detected a Spanish red table wine (Rioja) as a direct-acting mutagen. The best mutagenic response was obtained by preincubating strain BA13 with the wine sample in the presence of sodium phosphate buffer and in the absence of any external metabolic activation. In fact, the S9 mixture abolished most of the mutagenic activity of red wine in the Ara test. Such an inactivating capacity seems to be independent of microsomal enzymes and mediated through some kind of heat-stable component(s) in the S9 fraction. Both regular wine (directly from the bottle) and lyophilized wine were strong mutagens in the Ara test, inducing 4914 and 2739 AraR mutants/ml. Both pKM101 and delta uvrB were critical factors in the detection of the mutagenicity of wine, exhibiting a synergic effect in strain BA13. The mutagenicity of red wine was somehow pH-dependent, increasing with the pH value of the preincubation mixture. In comparison with the Ara test, the His reverse mutation assay (Ames test) was much less sensitive to the mutagenicity of lyophilized red wine, TA102 being the most (448 His+/ml) and TA98 the least (38 His+/ml) sensitive strain. TA100, TA104 and TA97 manifested intermediate mutagenicities to red wine. Previous reports have identified strain TA98 as the His strain most sensitive to the apolar fraction (e.g. XAD-2-bound) of red wine. Based on these results we propose that TA98 mainly detects glycosides of mutagenic flavonols present in red wine (quercetin, rutin, etc.), which do not constitute the major direct-acting mutagens detected with the Ara test.(ABSTRACT TRUNCATED AT 250 WORDS)

A forward and a reverse mutation assay designed to detect environmental mutagens have been compared in Salmonella typhimurium. The forward mutation assay scored resistance to L-arabinose and the reverse assay, reversion of histidine auxotrophy. Eighteen chemicals of different structural groups, all known to be mutagenic in the histidine reverse assay, were applied to strains carrying the genetic markers needed to perform both mutation assays. The mutagenicity of each chemical was determined by both plate and liquid tests. The plate test counted absolute numbers of surviving mutants and the liquid test separately measured survival and frequency of mutants among the survivors. All the chemicals used were found to be mutagenic in both mutation assays. The response of the L-arabinose assay was equal to or larger than the response of the histidine assay in the case of 16 chemicals. The two other compounds, 2-nitrofluorene and sodium azide, were detected more efficiently by the histidine assay. Sodium azide, a non-carcinogenic compound, is a potent mutagen in the histidine assay, but very weak in the L-arabinose assay.

This paper introduces a new parameter, derivable from dose-response data for induced mutagenesis in bacteria, that can be used to quantify mutational responses in short-term tests. We called this parameter the mutational response of the bipartite experimental system (agent plus cells). We defined it as being jointly proportional to the efficiency of the mutagen and the sensitivity of the test. We show how this quantity can be used to rank order chemical carcinogens on the basis of their mutagenicity and to determine the strength of any quantitative correlation that may exist between mutagenicity in bacteria and carcinogenicity in rodents. We find that this particular measure of mutational response for 10 direct-acting monofunctional alkylating agents correlates remarkably well with the rodent carcinogenicity of these chemicals measured in terms of their reciprocal TD50 values.