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

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

SOS-inducing activity of chemical carcinogens and mutagens in Salmonella typhimurium TA1535/pSK1002: examination with 151 chemicals

The umu test system is a newly developed method to evaluate genotoxic activities of a wide variety of environmental carcinogens and mutagens (Oda et al., 1985). In the present study, we further examined the abilities of 151 chemicals to induce umu gene expression in Salmonella typhimurium TA1535/pSK1002. Among the chemicals examined, 72 compounds induced umu gene expression, which could be defined on a basis of increased beta-galactosidase activity by 2-fold over the background level. The potent genotoxic compounds without metabolic activation were adriamycin, bleomycin, daunorubicin, 1,3-dinitropyrene, 1,6-dinitropyrene, 1,8-dinitropyrene, N-ethyl-N'-nitro-N-nitrosoguanidine, furylfuramide, methyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, mitomycin C, 1-nitropyrene and 4-nitroquino-line-1-oxide. In the presence of S9, aflatoxin B1, 2-aminoanthracene, Glu-P-1, IQ, MeIQ, MeIQx, Trp-P-1 and Trp-P-2 also induced umu gene expression markedly. Several chemicals such as 2-acetylaminofluorene, 9-aminoacridine, azobenzene, benzanthracene, benzidine, diethyl nitrosamine, 1-nitronaphthalene, paraquat, potassium dichromate and sodium nitrite were weakly genotoxic and the induction by these compounds could be detected only when the incubation time was prolonged from 2 h to 5 h. Data are also presented that some of the chemicals such as dimethyl sulfoxide, m-dioxan, 5-fluorouracil and paraquat, which have been reported to be non-mutagenic in Ames/Salmonella assay, were found to be active in inducing umu gene expression, while the known mutagenic compounds including acrylonitrile, 4,4'-dinitrobiphenyl, furfural, methylene chloride, 1-naphthylamine, sodium azide, o-tolidine and o-toluidine were non-genotoxic in the present assay system.     

Induction of umu gene expression in Salmonella typhimurium TA1535/pSK1002 by dimethyl sulfoxide (DMSO)

The genotoxicity of dimethyl sulfoxide (DMSO) was demonstrated by the umu test using Salmonella typhimurium TA1535/pSK1002 carrying the umuC-lacZ fusion gene. The level of beta-galactosidase activity which shows umu gene expression in the test system was dependent on the concentration of DMSO in the culture medium. The maximum beta-galactosidase activity was approximately 3.5 times as high as the background level with 10% of DMSO in the culture medium. The lowest concentration of DMSO required for a response of over twice the background level was approximately 5%. Four structurally related chemicals (acetone, di-n-butylsulfoxide, dimethylsulfide, methylphenylsulfoxide) did not show umu gene expression at their non-toxic doses.     

On the induction of umu gene expression in Salmonella typhimurium strain TA1535/pSK1002 by some nitrofurans.

Several nitrofurans were found to induce umu gene expression in Salmonella typhimurium TA1535/pSK1002 as defined on the basis of at least a 2-fold increase of beta-galactosidase activity over the background level. beta-Galactosidase activity increased with increasing concentrations of the chemical, attained a maximum at a concentration which was different for different nitrofurans used, and then gradually decreased with a further increase of the nitrofuran concentration. The umu gene expression test revealed that the genotoxic activity was highest for furazolidone and lowest for 5-nitro-2-furaldehyde.     

A microplate version of the SOS/umu-test for rapid detection of genotoxins and genotoxic potentials of environmental samples.

The umu-microtest is a miniaturized automated short-term test version proposed for screening of umuC-dependent mutagenic potentials of chemicals relevant to environmental pollution, river water and industrial waste water. The test is based on the SOS/umu-test and has been modified in order to allow extensive testing of environmental samples. Genetically engineered Salmonella typhimurium (TA1535/pSK1002) are incubated on a microplate rotor in a sloping position for 2 h with the test samples, followed by addition of fresh culture medium to reach a 10-fold dilution of the incubation medium. 2 h later, the activity of the beta-galactosidase, which reflects umuC induction, is determined colorimetrically. The incubation of the bacteria in the presence of the test compounds as well as the assessment of beta-galactosidase activity takes place in 96-well microplates, thus enabling simultaneous screening of large numbers of samples. Data of the genotoxic potentials are available within 8 h. Computer-controlled automation is possible by using a laboratory workstation.     

Electrochemical mutagen screening using microbial chip

Electrochemical microbial chip for mutagen screening were microfabricated and characterized by scanning electrochemical microscopy (SECM). Salmonella typhimurium TA1535 with a plasmid pSK1002 carrying a umuC'-'lacZ fusion gene was used for the whole cell mutagen sensor. The TA1535/pSK1002 cells were exposed to mutagen solutions containing 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamido (AF-2), mitomycin C (MMC) or 2-aminoanthracene (2-AA) and embedded in a microcavity (5nl) on a glass substrate using collagen gel. The beta-galactosidase expression on the microbial chip was electrochemically monitored using p-aminophenyl-beta-d-galactopyranoside (PAPG) as the enzymatic substrate. This system has several advantages compared with the conventional umu test: drastic reduction of the sample volume, less time-consuming for beta-galactosidase detection (free from substrate reaction time) and lower detection limit for the three mutagens (AF-2, MMC, 2-AA). Finally, a multi-sample assay was carried out using the microbial array chip with four microcavities.     

Metabolic activation of carcinogenic 1-nitropyrene by human cytochrome P450 1B1 in Salmonella typhimurium strain expressing an O-acetyltransferase in SOS/umu assay

Metabolic activation of 1-nitropyrene (1-NP) by human cytochrome P450 (P450) family 1 enzymes co-expressed with NADPH-cytochrome P450 reductase (NPR) in Escherichia coli membranes was investigated. 1-NP induced umu gene expression in Salmonella typhimurium TA1535/pSK1002 in the absence of any P450 system, but the activities were influenced by the levels of bacterial O-acetyltransferase (OAT) and nitroreductase. Metabolic activation of 1-NP by human P450 1B1/NPR membranes was observed and was influenced by the levels of OAT levels in tester strains. Metabolic activation of 1-NP (0.3microM) by P450 1B1 was 750 umu units/min/nmol P450 1B1 in an OAT-overexpressing strain NM2009. The metabolic activation of 1-NP (3-30microM) was similar (approximately 300 umu units/min/nmol P450 1B1) using TA1535/pSK1002 or OAT-deficient strain NM2000. P450 1B1 had the highest catalytic activities among P450 family 1 enzymes for the activation of 1-aminopyrene (1-AP) in the OAT-overexpressing strain NM2009, suggesting nitrenium ion formation via N-hydroxylation/O-acetylation. High-performance liquid chromatography (HPLC) analyses revealed the formation of 1-nitropyrene-6-ol and also 1-nitropyrene-3-ol, 1-nitropyrene-8-ol, and trans-4,5-dihydroxy-4,5-diol-1-nitropyrene from 1-NP (10microM), catalyzed by P450 1B1. These results indicate that 1-NP can be activated by human P450 1B1 to a genotoxic agent by nitroreduction/O-acetylation at low substrate concentrations and probably by epoxidation (independent of OAT) at high concentrations.     

Studies on the genotoxicity of endosulfan in bacterial systems

Endosulfan, an organochlorine pesticide, was subjected to the differential sensitivity assay in repair-deficient and repair-proficient strains of Escherichia coli K12, prophage lambda induction assay in WP2s (lambda) and mutation induction in E. coli K12. The induction of umu gene expression with endosulfan was studied also in Salmonella typhimurium TA1535/pSK1002 cells. The differential sensitivity assay revealed that the recA 13 strain was the most sensitive. Endosulfan induced prophage lambda in E. coli and umu gene expression in S. typhimurium cells; however, the extent of the effects were low. Endosulfan also induced a dose-dependent increase in forward mutations in E. coli K12 cells from ampicillin sensitivity to ampicillin resistance. Our studies indicate the genotoxic potential of endosulfan and the role of the recA gene in the repair of endosulfan-induced DNA damage.     

Development of a new genotoxicity test system with Salmonella typhimurium OY1001/1A2 expressing human CYP1A2 and NADPH-P450 reductase.

In order to develop a new tester strain detecting environmental promutagens and procarcinogens, we introduced two plasmids into Salmonella typhimurium TA1535; one contains the cDNAs of human cytochrome P450 (P450 or CYP) 1A2 and NADPH-P450 reductase and the other (pOA101) a umuC"lacZ fusion gene. The newly developed tester strain, S. typhimurium OY1001/1A2, was found to express P450 at a level of 0.15 nmol/ml in whole cell culture. Membrane fractions, when isolated from this tester strain, contained 0.04 P450 nmol/mg protein and a reductase activity of 170 nmol cytochrome c reduced/min/mg protein and were active in catalyzing CYP1A2-dependent 7-ethoxyresorufin O-deethylation and metabolic activation of heterocyclic aromatic amines to DNA-damaging products in a conventional tester S. typhimurium NM2009 strain, only when NADPH was added as a reducing equivalent. In the OA1002/1A2 strain, heterocyclic aromatic amines (e.g., IQ, MeIQ, and MeIQx) were found to be activated to reactive metabolites that cause induction of umuC gene expression in a dose-dependent manner, without addition of external NADPH. These results indicate that the newly established strain can be of use to detect mutagenic and carcinogenic potencies of environmental chemicals without addition of metabolic activation system.     

Antimutagenic activity of flavonoids from Chrysanthemum morifolium

A methanol extract from the flower heads of Chrysanthemum morifolium showed a suppressive effect on umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide). The methanol extract was re-extracted with hexane, chloroform, ethyl acetate, butanol, and water. The ethyl acetate fraction showed a suppressive effect. Suppressive compounds in the ethyl acetate fraction were isolated by silica gel column chromatography and identified as the flavonoids acacetin (1), apigenin (2), luteolin (3), and quercetin (4) by EI-MS, IR, and (1)H and 13C NMR spectroscopy. Compounds 1-4 suppressed the furylfuramide-induced SOS response in the umu test. Compounds 1-4 suppressed 60.2, 75.7, 90.0, and 66.6% of the SOS-inducing activity at a concentration of 0.70 micromol/ml. The ID50 (50% inhibitory dose) values of 1-4 were 0.62, 0.55, 0.44, and 0.59 micromol/ml. These compounds had the suppressive effects on umu gene expression of the SOS response against other mutagens, 4-nitroquinolin 1-oxide (4NQO) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which do not require liver-metabolizing enzymes. These compounds also showed the suppression of SOS-inducing activity against the other mutagens aflatoxin B1 (AfB1) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), which require liver-metabolizing enzymes, and UV irradiation. In addition to the antimutagenic activities of these compounds against furylfuramide, Trp-P-1 and activated Trp-P-1 were also assayed by the Ames test using S. typhimurium TA100.     

Revised methods for the Salmonella mutagenicity test

The methods for detecting carcinogens and mutagens with the Salmonella mutagenicity test were described previously (Ames et al., 1975b). The present paper is a revision of the methods. Two new tester strains, a frameshift strain (TA97) and a strain carrying an ochre mutation on a multicopy plasmid (TA102), are added to the standard tester set. TA97 replaces TA1537. TA1535 and TA1538 are removed from the recommended set but can be retained at the option of the investigator. TA98 and TA100 are retained. We discuss other special purpose strains and present some minor changes in procedure, principally in the growth, storage, and preservation of the tester strains. Two substitutions are made in diagnostic mutagens to eliminate MNNG and 9-aminoacridine. Some test modifications are discussed.     

6-Dimethylaminopurine (6-DMAP), which is used to produce most cloned animals, is mutagenic in Salmonella typhimurium TA1535

6-Dimethylaminopurine (6-DMAP) and cycloheximide have recently been used for successful production of cloned mammals. We investigated whether 6-DMAP or cycloheximide are mutagenic agents in the Ames test. Whereas cycloheximide showed no differences compared to the negative control in any of the tester strains, 6-DMAP was clearly mutagenic in Salmonella typhimurium strain TA1535. Here, we strongly propose that innocuous chemicals be used in the production of cloned animals.     

The lipid peroxidation product 4-hydroxynonenal is a potent inducer of the SOS response.

An important aspect of bacterial mutagenesis by several difunctional carbonyl compounds appears to be the induction of the SOS system. We tested the ability of a series of carbonyl compounds to induce expression of the SOS-regulated umu operon in Salmonella typhimurium TA1535/pSK1002. SOS-inducing potencies varied widely among the carbonyl compounds tested. 4-Hydroxynonenal, a product of lipid peroxidation, was the most potent SOS-inducer, with maximal induction observed at concentrations of 0.1-1 microM. Acrolein, crotonaldehyde and methacrolein induced little increase over background umu expression. Malondialdehyde, another product of lipid peroxidation, was a very weak SOS-inducer with a maximal response induced at a concentration of 28 mM. Substitution at the alpha-position of malondialdehyde, which abolishes frameshift mutagenicity, did not abolish SOS-inducing activity. Substitution of the hydroxyl group of malondialdehyde and alpha-methyl-malondialdehyde by a better leaving group (benzoyloxy) resulted in an approximately 250-fold higher SOS-inducing potency. Comparison of the present results to literature reports on bacterial mutagenicity indicates a poor correlation of the two properties between different classes of difunctional carbonyl compounds and even within the same class of difunctional carbonyl compounds.     

6-Dimethylaminopurine (6-DMAP), which is used to produce most cloned animals, is mutagenic in Salmonella typhimurium TA1535

6-Dimethylaminopurine (6-DMAP) and cycloheximide have recently been used for successful production of cloned mammals. We investigated whether 6-DMAP or cycloheximide are mutagenic agents in the Ames test. Whereas cycloheximide showed no differences compared to the negative control in any of the tester strains, 6-DMAP was clearly mutagenic in Salmonella typhimurium strain TA1535. Here, we strongly propose that innocuous chemicals be used in the production of cloned animals.     

Curing and induction of the Fels 1 and Fels 2 prophages in the Ames mutagen tester strains of Salmonella typhimurium

A method is described for curing the Ames Salmonella mutagen tester strains of their Fels 1 and Fels 2 prophages with the aid of the antitumor drug daunorubicin. Non-lysogenic derivatives corresponding to TA100 and TA1535 were isolated and designated TAQ100 and TAQ1535 respectively. In addition, the Fels 1 monolysogens TAQ100F1 and TAQ1535F1, as well as the Fels 2 monolysogens TAQ100F2 and TAQ1535F2, were obtained. Finally, strains corresponding to TA98 and TA1538 cured of Fels 2, but retaining a cryptic Fels 1 (F1d) prophage were isolated and designated TAQ98F1d and TAQ1538F1d respectively. The various cured derivatives were identified by colony hybridization with ³²P-labeled probes of Fels 1 and Fels 2 DNA. Southern blot hybridizations confirmed that phage-specific Fels DNA sequences were missing from the cured strains. The Fels 2-cured strains were resistant to Fels 2, but Fels 1 grew, albeit poorly, on the Fels 1-cured strains. Strains TAQ100F1, TAQ1535F1, TAQ100F2 and TAQ1535F2 were used in prophage induction assays, in the presence of rat-liver extract where necessary. Daunorubicin, bleomycin, mitomycin C, aflatoxin B1, 2-amino-dipyrido[1,2-a:3′,2′-d]imidazole (Glu-P-2) were found to induce Fels 1 and/or Fels 2 in at least one of these strains. The induction of the Fels prophages in the TAQ monolysogens may provide a useful complement to the Ames test for the detection of DNA-damaging agents and potential carcinogens.     

Genotoxic activation of benzophenone and its two metabolites by human cytochrome P450s in SOS/umu assay

The genotoxic potential of benzophenone and its metabolically related compounds, benzhydrol and p-benzoylphenol, was investigated using human cytochrome P450 (P450) enzymes. Benzophenone and its two metabolites (0.1-1mM) showed a suppression of bacterial growth without any P450 system, but no induction of umu gene expression was observed in Salmonella typhimurium TA1535/pSK1002. Human liver microsomes induced the bacterial cytotoxicity of these compounds without any umu gene expression. On the other hand, with the addition of Escherichia coli membranes expressing recombinant human P450 2A6 and NADPH-cytochrome P450 reductase (NPR), benzophenone showed umu gene expression (64 umu units/min/nmol) P450 2A6). Moderate activation of benzophenone by P450 1A1/NPR membranes, 1A2/NPR membranes, or 1B1/NPR membranes was also observed. Activation of benzhydrol and p-benzoylphenol by the P450/NPR system was similar to that of benzophenone. These results suggest that benzophenone and its metabolically related benzhydrol and p-benzoylphenol can be bioactivated by P450 2A6 and P450 family 1 enzymes. Until now, benzophenone has been investigated mainly in terms of estrogenic activity and cytotoxicity, however, the genotoxic activation of benzophenone by human cytochrome P450s should be examined in terms of the risks to humans.     

Induction of umu gene expression by cross-links and other DNA lesions

The induction of umu gene expression by DNA cross-links was investigated in various strains of E. coli with different DNA-repair capacities. Expression was measured by quantifying enzymatic activity of beta-galactosidase produced under regulation of the umu promoter carried on a plasmid carrying the umuC-lacZ gene fusion. The treatment with MMC induced gene expression more efficiently in a wild-type strain when compared with an excision-repair-deficient strain (uvrA). In contrast, PUVA and cis-Pt treatment induced higher levels of the gene expression in the uvrA strain than in the wild-type strain, as did other DNA-damaging agents including 4NQO, MNNG and MMS. None of these chemicals induced umu expression in either lexA and recA strains. The mechanisms of the induction of umu expression by DNA cross-links in relation to DNA damage and repair are discussed.     

Response of the L-arabinose forward mutation assay of Salmonella typhimurium to frameshift-type mutagens

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.     

Plasmid pSK1002-mediated mutator effect and SOS response and SOS mutagenesis of 2,5-dichloronitrobenzol in Salmonella typhimurium

The plasmid pSK1002 (umuC'-'lacZ) could increase the number of revertants induced by methyl methanesulfonate (MMS) and 4-nitroquinoline-N-oxide (4NQO) in Salmonella typhimurium TA 1535 (his-). The values induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were not different irrespective of the presence or absence of plasmid. However, the plasmid pKM101-mediated mutagenesis-enhancing effect was much greater than that mediated by pSK1002 as induced by the 3 mutagens mentioned above. Moreover, the plasmid pSK1002 could induced umu-mediated SOS response in the presence of any of these 3 mutagens or of mitomycin C, and a dose-response relationship was evident. It shows that pSK1002 (umuC'-'lacZ) has a dual biological effect, namely a mutator effect and the effect of inducing the SOS response. Besides, this study has proved SOS mutagenesis of 2,5-dichloronitrobenzol (2,5-DCNB) because of the dual indicator nature of pSK1002. Therefore, it is probable that pSK1002 could be further developed and applied in studying the relation between the SOS response and mutagenesis and in identifying environmental SOS mutagens.     

Facile synthesis of 4-O-β-N-Acetylchitooligosyl 2-acetamido-2,3-dideoxydidehydro-gluconolactone based on the transformation of chitooligosaccharide and its suppressive effects against the furylfuramide-induced SOS response

A facile synthesis method is described for transforming the reducing-end residue of chitooligosaccharides (DP 2-4) into lactone. The desired 4-O-β-N-acetylchitooligosyl lactones (GN(n)L) were conveniently prepared from chitooligosaccharides by consecutive dehydration and oxidation reactions to afford 4-O-β-tri-N-acetylchitotriosyl 2-acetamido-2,3-dideoxydidehydro-gluconolactone (GN(3)L), 4-O-β-di-N-acetylchitobiosyl 2-acetamido-2,3-dideoxydidehydro-gluconolactone (GN(2)L), and 4-O-β-2-acetamido-2-deoxy-D-glucopyranosyl 2-acetamido-2,3-dideoxydidehydro-gluconolactone (GNL). The resulting lactone derivatives exhibited considerable suppression (42.6-54.3% at a concentration of 400 μM) in umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamido (AF-2). Lactonization of the chitooligosaccharides was found to be essential for their suppression of the SOS-inducing activity.