Comparative bacterial mutagenicity studies with 8-methoxypsoralen and 4,5′,8-trimethylpsoralen in the presence of near-ultraviolet light and in the dark

2 strains of S. typhimurium, TA98 and TA100, and 2 strains of E. coli, WP2(pKM101) and WP2uvrA⁻(pKM101) were used to study mutagenesis by 8-methoxypsoralen (8-MOP) and 4,5′,8-trimethylpsoralen (4,5′,8-TMP) in the dark and in the presence of near-ultraviolet (NUV) light both without metabolic activation and with rat-liver S9 at 3 levels (4, 10 and 30% in standard cofactors).The S9-independent base substitution mutagenic activity of 8-MOP plus NUV light was confirmed in WP2(pKM101), and a similar activity was seen for 4,5′,8-TMP, although neither substance was active in TA100. The frameshift mutagenic activity of 8-MOP in the dark in TA98 was not confirmed despite histidine levels which would ensure DNA replication, but this may be due to the lower concentrations of 8-MOP achieved in the common solvent system adopted.Both 8-MOP and 4,5′,8-TMP were mutagenic in WP2uvrA⁻(pKM101) after microsomal activation, and the responses were similar whether experiments were conducted in the dark or in NUV light. In view of the oral administration of 8-MOP to psoriasis patients, this finding may be of relevance in risk assessment, and tends to suggest that topical application of 4,5′,8-TMP to psoriatic patients may present reduced risk of malignant disease.     

Mutagenic activity of furfural in Salmonella typhimurium TA100

The mutagenic activity of furfural was tested in Salmonella typhimurium strains TA98 and TA100. Furfural produced mutations in the TA100 strain, but not in the TA98 strain. A rat-liver microsomal fraction did not increase the mutagenic activity of furfural in either strain. Mutagenic activity of furfural in the TA100 strain was not increased by benzo[a]pyrene in the presence of metabolic activation.     

Alteration of bacteriophage attachment capacity by near-UV irradiation.

Near-UV (NUV) (300 to 400 nm) and far-UV (FUV) (254 nm) radiations damage bacteriophage by different mechanisms. Host cell reactivation, Weigle reactivation, and multiplicity reactivation were observed upon FUV, but not upon NUV irradiation. Also, the number of his+ recombinants increased with P22 bacteriophage transduction in Salmonella typhimurium after FUV, but not after NUV irradiation. This loss of reactivation and recombination after NUV irradiation was not necessarily due to host incapability to repair phage damage. Instead, the phage genome failed to enter the host cell after NUV irradiation. In the case of NUV-irradiated T7 phage, this was determined by genetic crosses with amber mutants, which demonstrated that either "all" or "none" of a T7 genome entered the Escherichia coli cell after NUV treatment. Further studies with radioactively labeled phage indicated that irradiated phage failed to adsorb to host cells. This damage by NUV was compared with the protein-DNA cross-link observed previously, when phage particles were irradiated with NUV in the presence of H2O2. H2O2 (in nonlethal concentration) acts synergistically with NUV so that equivalent phage inactivation is achieved by much lower irradiation doses.     

Non-enzymic activation of polycyclic aromatic hydrocarbons as mutagens.

Samples of 22 polycyclic aromatic hydrocarbons and related derivatives were subjected to ⁶⁰Co gamma radiation in air, and the irradiated samples were tested for mutagenicity with the Salmonella typhimurium strains TA 98, TA 1535, TA 1537, and TA 1538. Testing was conducted with the bacterial strains alone, thus not fortified with liver-microsomal enzymes or other metabolizing systems. Marked mutagen responses were obtained for several irradiated samples with the TA 98, TA 1537, and TA 1538 strains but not with the TA 1535 strain. Irradiated samples of benzo[a]anthracene, benzanthrone, benozo[g,h,i]perylene, benzo[a]pyrene, chrysene, fluorene, 9-methylanthracene, 1-methylphenanthrene, 2-methylphenanthrene, and pyrene gave positive mutagenic tests and dose-responses, whereas unirradiated control samples of these were inactive. Acenaphthene, phenanthrene, and phenanthrenequinone exhibited toxicity which interfered with interpretation of mutagenicity testing. Samples of 2-methylanthracene and tetracene were mutagenic with or without irradiation. Alizarin, anthracene, anthraquinone, anthrone, dobenzo[a,h]anthracene, picene, and triphenylene negative results. Samples of benzo[a]pyrene adsorbed on silica gel irradiated in air by ⁶⁰Co gamma radiation or by 254 nm ultraviolet light and samples adsorbed on filter paper irradiated by visible light yielded preparations mutagenic towards the TA 98, TA 1537, and TA 1538 strains. These results suggest that parent polycyclic aromatic hydrocarbons not themselves mutagenic towards S. typhimurium may be oxidized in air by radiation-induced processes to products whose mutagenicity resembles that of liver-microsomal metabolites of the parent polycyclic aromatic hydrocarbon.     

Evaluation of the mutagenic potential of mycotoxins using Salmonella typhimurium and Saccharomyces cerevisiae

The mutagenic effects of fifteen mycotoxins on Salmonella typhimurium strains TA1535, TA1537 and TA1538 and Saccharomyces cerevisiae strain D-3 were tested. Only aflatoxin B₁ and sterigmatocystin were mutagenic. Both were active against S. typhimurium strain TA1538 and S. cerevisiae strain D-3; however, both required activation by the hepatic S-9 enzyme preparation. A positive correlation between the other mycotoxins reported to be carcinogenic and the two in vitro test systems employed was not demonstrated in our hands.     

Comparative mutability of the Ames tester strains of Salmonella typhimurium by ultraviolet radiation and by 4-nitroquinoline 1-oxide

A standard method for determining mutant frequencies per survivor was used to study the detailed kinetics of reverse mutations of Ames tester strains of Salmonella typhimurium induced by UV and by 4N1O. After UV irradiation, strain TA1538 was non-mutable, but its plasmid-containing derivative TA98 was mutable, whereas TA1535 was mutable and its plasmid-bearing derivative TA100 was about 10-fold more mutable. After treatment with 4NQO, TA98 was less mutable than TA1538, whereas TA100 was more mutable than TA1535 by a factor of 10–50. TA1537 was slightly less mutable than TA1535 by either UV or 4NQO. The differential mutabilities of these strains are briefly discussed in relation to the “hot spot” base sequences for reversion and the nature of DNA damage caused by UV and 4NQO.     

Mutagenesis by photoaffinity labeling using selected azidofluorenes

Several 2-azidofluorenes have been synthesized for use as photoaffinity labels inside bacteria. In the dark they were not mutagenic for any Salmonella typhimurium tested. When photolyzed inside the bacteria, all were mutagenic for strain TA1538 to varying degrees, and were considerably less mutagenic in the corresponding repair positive TA1978. None were mutagenic for strain TA1535 or TA1537, although most compounds were toxic for those strains when photolyzed.     

Fluroxene mutagenicity

The commercially available volatile anesthetic fluroxene (2,2,2-trifluoroethyl vinyl ether) which contains the stabilizer N-phenyl-1-napthylamine, was tested for mutagenicity using four strains of S. typhimurium, TA1535, TA1537, TA98 and TA100, and one strain of E. coli, WP2. In addition, purified fluroxene; N-phenyl-1-napthylamine; trifluoroethanol, a major metabolite of fluoroxene; and urine from rats anesthetized with fluroxene were tested. Several procedures were utilized including exposure of bacteria to vapor in desiccators and in liquid suspension. Results indicate that fluroxene, but not its stabilizer, was mutagenic to strains TA1535, TA100 and WP2 only in liquid suspension and only in the presence of a rat-liver enzyme system. Trifluoroethanol and urine from fluroxene-treated rat were not mutagenic to any strain of bacteria. These findings indicate that fluroxene is a promutagen which requires preincubation before it is recognized. Further experiments were performed with enzymes prepared from mouse, hamster and human liver. Fluroxene was mutagenic only in the presence of enzymes prepared from Aroclor 1254 pretreated rodents. Since fluroxene was not mutagenic in the presence of enzymes prepared from three human livers, the significance of these findings to man are unclear.     

Comparisons of mutation induction in reversion systems of Saccharomyces cerevisiae and Salmonella typhimurium

The principle of the treatment condition routinely used in Salmonella typhimurium is to allow the cells to divide in the presence of the chemical being tested; only the revertants will be able to form visible colonies (softagar procedure). In Saccharomyces cerevisiae, the routinely used procedure is to treat the cells in liquid non-nutrient medium under non-growing conditions (non-nutrient procedure). We compared mutation induction under both experimental conditions using S. cerevisiae; we also compared the mutagenic response of the two microorganisms to six compounds; two nitrofuran derivatives, AF-2 and SQ18,506, three hair dye components, 1,2-diamino-4-nitrobenzene, 1,4-diaminoanthraquinone, and methyl violet, as well as ethyl methanesulfonate. Of the six compounds tested in S. cerevisiae strain XV185-14C, only ethyl methanesulfonate was mutagenic under both experimental conditions. The two nitrofuran derivatives, AF-2 and SQ18,506, induced mutations in S. cerevisiae when the non-nutrient procedure was employed. None of the three hair dyes tested was mutagenic in S. cerevisiae. However, the results obtained with Salmonella typhimurium indicate that the hair dye 1,2-diamino-4-nitrobenzene is a mutagen, confirming the earlier study by Ames et al. [2]. Among the other five compounds tested in Salmonella typhimurium, the base-substitution-detecting strain TA100 responded to one concentration of AF-2, and EMS was mutagenic in strains TA1535, TA100 and TA1537.     

Sensitivity of Deinococcus radiodurans to near-ultraviolet radiation

Although Dienococcus radiodurans is notoriously resistant to far-ultraviolet radiation (FUV; 254 nm), it is highly sensitive to near-ultraviolet radiation (NUV; 300-400 nm), thus demonstrating that the mechanisms of damage (and/or recovery) by the two types of irradiation are different. This observed difference between FUV and NUV effects in D. radiodurans agrees with previous studies with Escherichia coli. Near-ultraviolet radiation produces DNA damage which is presumed to be single-strand breaks (SSB) in the DNA of D. radiodurans. Unique lesions, such as DNA-protein crosslinks could not be demonstrated in this study. Cells that were pre-irradiated with a small dose of NUV were subsequently protected against inactivating doses of NUV. The data presented are consistent with induced DNA repair following NUV damage in D. radiodurans; this is in contrast to FUV damage where DNA repair is constitutive but not induced.     

Mutagenicity of aliphatic epoxides

The mutagenicity of 17 aliphatic epoxides was determined using the specially constructed mutants of Salmonella typhimurium developed by Ames. The activity of these epoxides together with those reported in the literature as mutagens in strains TA100 and TA1535 depended on the degree of substitution around the oxirane ring. Monosubstituted oxiranes were the most potent mutagens in both strains. 1,1-Disubstitution resulted in the complete loss or reduction of mutagenicity. trans-1,2-Disubstituted, and tetrasubstituted oxiranes all lacked mutagenicity, while the cis-1,2-disubstituted oxiranes tested were weakly mutagenic in strain TA100 only. For the monosubstituted compounds the presence of electron-withdrawing substituents increased mutagenicity.     

Mutation of Chinese hamster cells by near-UV activation of promutagens

A tissue-culture assay for mutagenesis and cytotoxicity incorporating near ultraviolet (NUV) light activation of polyaromatic hydrocarbons (PAH) has been developed. Cultures of Chinese hamster cells (line CHO) growing in suspension culture were inoculated with benzo[a]pyrene (B[a]P), 7,12-dimethylbenzanthracene (DMBA) or shale-oil retort-water and exposed to light from a high-pressure mercury lamp fitted with a Corning NUV bandpass filter. This light source both permitted activation of PAH and the shale-oil water and preculded detectable damage to DNA. Neither the PAH nor the NUV alone had any effect on cell survival or mutation frequencies but the chemicals plus NUV were extremely effective in producing mutations to 6-thioguanine resistance (hgprt gene).     

Comparative bacterial mutagenicity studies with 8-methoxypsoralen and 4,5',8-trimethylpsoralen in the presence of near-ultraviolet light and in the dark

2 strains of S. typhimurium, TA98 and TA100, and 2 strains of E. coli, WP2(pKM101) and WP2uvrA-(pKM101) were used to study mutagenesis by 8-methoxypsoralen (8-MOP) and 4,5',8-trimethylpsoralen (4,5',8-TMP) in the dark and in the presence of near-ultraviolet (NUV) light both without metabolic activation and with rat-liver S9 at 3 levels (4, 10 and 30% in standard cofactors). The S9-independent base substitution mutagenic activity of 8-MOP plus NUV light was confirmed in WP2(pKM101), and a similar activity was seen for 4,5',8-TMP, although neither substance was active in TA100. The frameshift mutagenic activity of 8-MOP in the dark in TA98 was not confirmed despite histidine levels which would ensure DNA replication, but this may be due to the lower concentrations of 8-MOP achieved in the common solvent system adopted. Both 8-MOP and 4,5',8-TMP were mutagenic in WP2uvrA-(pKM101) after microsomal activation, and the responses were similar whether experiments were conducted in the dark or in NUV light. In view of the oral administration of 8-MOP to psoriasis patients, this finding may be of relevance in risk assessment, and tends to suggest that topical application of 4,5',8-TMP to psoriatic patients may present reduced risk of malignant disease.     

Construction of a Symbiotically Effective Strain of Rhizobium leguminosarum bv. trifolii with Increased Nodulation Competitiveness

Genes involved in nodulation competitiveness (tfx) were inserted by marker exchange into the genome of the effective strain Rhizobium leguminosarum bv. trifolii TA1. Isogenic strains of TA1 were constructed which differed only in their ability to produce trifolitoxin, an antirhizobial peptide. Trifolitoxin production by the ineffective strain R. leguminosarum bv. trifolii T24 limited nodulation of clover roots by trifolitoxin-sensitive strains of R. leguminosarum bv. trifolii. The trifolitoxin-producing exconjugant TA1::10-15 was very competitive for nodulation on clover roots when coinoculated with a trifolitoxin-sensitive reference strain. The nonproducing exconjugant TA1::12-10 was not competitive for nodule occupancy when coinoculated with the reference strain. Tetracycline sensitivity and Southern analysis confirmed the loss of vector DNA in the exconjugants. Trifolitoxin production by TA1::10-15 was stable in the absence of selection pressure. Transfer of tfx to TA1 did not affect nodule number or nitrogenase activity. These experiments represent the first stable genetic transfer of genes involved in nodulation competitiveness to a symbiotically effective Rhizobium strain.     

Mutagenicity of glyceryl trinitrate (nitroglycerin) in Salmonella typhimurium

The recent finding that the clinical nitrovasodilator, glyceryl trinitrate (GTN), is mutagenic in Salmonella typhimurium strain TA1535 has been examined in closer detail, with emphasis on its mechanism of action. GTN increased the number of His⁺ revertants to a maximum of 4 times over background at a GTN dose of 5 μmol/plate. Hamster liver S9 depressed the toxicity of high GTN doses and increased the maximum number of revertants to 5 times over background at 10 μmol/plate. GTN did not cause significant reversion in any of the six other S. typhimurium strains tested (TA1975, TA102, TA1538, TA100, TA100NR, YG1026), although signs of toxicity were observed. Therefore, the mutagenicity of GTN was manifest only in the repair-deficient (uvrB and lacking in pKM101) strain which is responsive to single base changes. Oligonucleotide probe hybridization of TA1535 revertants showed that virtually all of the GTN-induced mutants contained C → T transitions in either the first or second base of the hisG46 (CCC) target codon, with a preference for the latter. A similar mutational spectrum was seen previously with a complex of spermine and nitric oxide (NO) which releases nitric oxide. This suggests that NO, which can be derived from GTN via metabolic reduction, may be responsible for GTN's mutagenic action. The known NO scavenger oxymyoglobin did not substantially alter the dose response of GTN, indicating that extracellular NO was not mediating reversion. The data are consistent with the hypothesis that intracellular nitric oxide is responsible for the observed mutations.     

Mutagenicity of flavones,chromones and acetophenones in Salmonella typhimurium: New structure—activity relationships

28 flavones and 11 structurally-related flavonoids, chromones, and acetophenones, were tested for mutagenicity in the Salmonella typhimurium his reversion assay. 7 flavones, all of which were hydroxy- or methoxy-substituted at position 8, were moderate to strong mutagens in strain TA100 in the presence of rat liver S9 mix. In each case, the response of strain TA98 was either not significant or was very much weaker than that observed in strain TA100. The activation by S9 is not mediated by the microsomal cytochrome P₄₅₀ system, since activation was not diminished when microsomes were removed by centrifugation at 100000 × g. The observed strain specificity and structural requirements for activity indicate a mutagenic mechanism different from that associated with previously reported mutagenic flavonols (3-hydroxy-flavones) which are most active in strain TA98. The most mutagenic flavone investigated, 5,7,8-trihydroxy-flavone (norwogonin), had a potency of 17 revertants/nmole.Simplification of the chemical structures to hydroxy-substituted chromone and acetophenone systems revealed similar strain specificity, hydroxylation requirements, and S9 dependence within these structural classes, suggesting a similar activation pathway and mutagenic mechanism. The greatest mutagenic potency was observed within the flavone series, but significant potency was retained by similarly hydroxylated chromones and acetophenones. No mutagenic activity was observed in the absence of the aryl ketone moiety.     

Cytotoxic effects of norethindrone-4β,5β-epoxide to Walker cells in culture and to rat liver in vivo

Selected platinum and ruthenium complexes were tested for their ability to cause Salmonella typhimurium strains TA98 and TA100 to revert to histidine independence. The results indicate that ruthenium compounds are capable of reverting both strains while cis-Cl₂(NH₃)₂Pt primarily causes reversions in strain TA100. In addition, cis-platinum is an order of magnitude more mutagenic and toxic than are the ruthenium complexes. Selected compounds were also tested for their ability to induce the bacterial SOS system in the Bacillus subtilis Comptest. In this system, cis-platinum similarly showed greater inducing ability than did the ruthenium complexes. These results also demonstrated that the nature of the sixth ligand in the ruthenium compounds has a significant effect on the mutagenic capacity of these agents.     

Toxicity and Mutagenicity of Hexavalent Chromium on Salmonella typhimurium

Four hexavalent and two trivalent chromium compounds were tested for toxicity and mutagenicity by means of the Salmonella typhimurium/mammalian-microsome test. All hexavalent compounds yielded a complete inhibition of bacterial growth at doses of 400 to 800 μg/plate, a significant increase of his⁺ revertant colonies at doses ranging from 10 to 200 μg, and no effect at doses of less than 10 μg. The distinctive sensitivity of the four Salmonella strains tested (TA1535, TA1537, TA98, and TA100) suggested that hexavalent chromium directly interacts with bacterial deoxyribonucleic acid by causing both frameshift mutations and basepair substitutions. The latter mutations, which are prevalent, are amplified by an error-prone recombinational repair of the damaged deoxyribonucleic acid. On the average, 1 μmol of hexavalent chromium yielded approximately 500 revertants of the TA100 strain, irrespective of the compound tested (sodium dichromate, calcium chromate, potassium chromate, or chromic acid). The mutagenic potency of the hexavalent metal was not enhanced by adding the microsomal fraction of rat hepatocytes, induced either with sodium barbital or with Aroclor 1254. The two trivalent compounds (chromium potassium sulfate and chromic chloride), with or without the microsomal fraction, were neither toxic nor mutagenic for the bacterial tester strains.     

In vitro genotoxicity of neutral red after photo-activation and metabolic activation in the Ames test, the micronucleus test and the comet assay

Neutral red (Nr) is relatively non-toxic and is widely used as indicator dye in many biological test systems. It absorbs visible light and is known to act as a photosensitizer, involving the generation of reactive oxygen species (type-I reaction) and singlet oxygen (type-II reaction). The mutagenicity of Nr was determined in the Ames test (with Salmonella typhimurium strains TA1535, TA97, TA98, TA98NR, TA100, and TA102) with and without metabolic activation, and with and without photo-activation on agar plates. Similarly to the situation following metabolic activation, photo-mutagenicity of Nr was seen with all Salmonella strains tested, albeit with different effects between these strains. To our knowledge, Nr is the only photo-mutagen showing such a broad action. Since the effects are also observed in strains not known to be responsive to ROS, this indicates that ROS production is not the sole mode of action that leads to photo-genotoxicity. The reactive species produced by irradiation are short-lived as pre-irradiation of an Nr solution did not produce mutagenic effects when added to the bacteria. In addition, mutagenicity in TA98 following irradiation was stronger than in the nitroreductase-deficient strain TA98NR, indicating that nitro derivatives that are transformed by bacterial nitroreductase to hydroxylamines appear to play a role in the photo-mutagenicity of Nr. Photo-genotoxicity of Nr was further investigated in the comet assay and micronucleus test in L5178Y cells. Concentration-dependent increases in primary DNA damage and in the frequency of micronuclei were observed after irradiation.