Suppression of mutagens-induced SOS response by phytoncide solution using Salmonella typhimurium TA1535/pSK1002 umu test.

Four types of phytoncide solution (A-Type, AB-Type, D-Type and G-Type) were evaluated as antimutagenic agents with suppressive effects on the SOS-inducing activity of the mutagen 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (furylfuramide) using Salmonella typhimurium TA1535/pSK1002 umu test. The A-Type, AB-Type, D-Type and G-Type of phytoncide solution suppressed the SOS-inducing activity on furylfuramide at a concentration of 100 microg/mL by 86.1%, 74.7%, 69.5% and 55.4%, respectively, and the ID(50) (50% inhibitory dose) values were 9.0 microg/mL, 22.5 microg/mL, 36.0 microg/mL and 72.8 microg/mL. They also showed the suppression of SOS-inducing activity against other chemical mutagens, such as 4-nitroquinolin 1-oxide (4NQO) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which do not require liver metabolizing enzymes, and against 2-aminoanthracene (2AA) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), which require these enzymes, and against UV irradiation, which is a well known physical mutagen. In the search for the component-activity relationship, the A-Type of phutoncide solution suppressed the SOS-inducing activity greater than the other types of phutoncide solution for furylfuramide, 4NQO and MNNG. However, in case of 2AA and Trp-P-1, the D-Type of phytoncide solution was most effective in suppressing the SOS-inducing activity in the umu test. From these results, the four types of phytoncide solutions showed the suppressive effect of SOS-inducing activity against chemical and physical mutagens.

Antimutagenic potential of the Thai herb, Mucuna collettii Lace.

Mucuna collettii Lace is a Thai herb with a long record of consumption among mature Thai males for the promotion of sexual potency. The mutagenic and antimutagenic potentials of Mucuna collettii extract were carried out by using the Ames test pre-incubation method in the presence and absence of S9 mixture. Salmonella typhimurium strains TA 98 and TA 100 were applied as the tester strains. Prior to mutagenic and antimutagenic tests, the survival of the tester strains was performed by treating with the plant extract. Results showed Mucuna collettii extract exhibited strong cytotoxic effects in a dose-dependent manner. Toxicity of the plant was confirmed in mice in which negative adverse effect was found in kidney, uterus, ovary, and testis. Mucuna collettii extract in the presence and absence of S9 mixture was negative for mutagenic Ames test. Mucuna collettii extract in the presence and absence of S9 mixture was positive for antimutagenic Ames test towards either one or both of the tested mutagens: 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF-2) and benzo(a)pyrene. The antimutagenic activity of the plant extract was confirmed in rec-assays. Micronucleus test demonstrated that Mucuna collettii extract at high dose and a long incubation time could induce micronucleus formation in tested animals, but less than the response of the positive control. The overall mutagenic and antimutagenic assays are further evidences for the antimutagenic potential of Mucuna collettii.

Effects of nine active ingredients in Chinese herbal medicine sho-saiko-to on 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide mutagenicity.

The antimutagenic effects of nine active compounds in the Chinese herbal medicine "sho-saiko-to" on mutagenesis induced by a direct-acting mutagen, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) were investigated in Salmonella typhimurium, strain TA100. The active compounds examined were classified into two major groups, saponins and flavonoids, the former comprising glycyrrhizin, saikosaponins a, c, and d, and ginsenosides Rb1 and Rg1, and the latter, baicalin, baicalein and wogonin. Saikosaponin a and ginsenoside Rb1 were found to reduce the mutagenicity of AF-2 significantly when applied post-AF-2-treatment in the Salmonella mutagenicity assay. Ginsenoside Rb1 also decreased the mutagenic activity of AF-2 in a simultaneous treatment protocol. The results indicate that saikosaponin a and ginsenoside Rb1 may enhance DNA repair, and ginsenoside Rb1 may also have the ability to inactivate the mutagenic activity of AF-2 directly. On the other hand, saikosaponin d and baicalin showed a slight enhancing effect. None of the compounds, except baicalein, showed any toxic effect on the test strain. These findings may be useful for the development of chemopreventive agents.

DNA fingerprint analysis in chemically mutagenized Chinese hamster lung cells.

Using a multi-locus minisatellite Per-6 DNA probe, we performed DNA fingerprint analysis. Chinese hamster lung (CHL) cells were treated with six model chemicals: N-methyl-N'-nitro-N-nitrosoguanidine, mitomycin C, methyl methanesulfonate, furylfuramide, 2-acetylamino-fluorene, and cyclophosphamide, with or without S9 mix. 771 hypoxanthine phosphoribosyltransferase deficient clones (749 from mutagen-treated cells and 22 from untreated cells) and 90 unselected clones from untreated cells were isolated and analyzed. The spontaneous mutation frequency at CHL cell minisatellite loci was 0.31-0.63%. All the chemicals increased mutation frequencies. Almost all mutations localized to the three specific minisatellite loci corresponding to 4.2, 3.8, and 2.4 kb bands, suggesting that these regions are more unstable and susceptible to mutation. DNA fingerprint analysis is a promising technique for detecting mutations at neutral DNA regions, especially recombinational mutations, and may be useful for surveying genetic instability related to heritable defects or aging.

Effects of the umuDC, mucAB, and samAB operons on the mutational specificity of chemical mutagenesis in Escherichia coli: I. Frameshift mutagenesis.

The specificity of frameshift mutations induced by several classes of chemical mutagens was determined using a collection of mutant E. coli lacZ genes. This collection can detect each of five kinds of specific frameshift events by scoring Lac+ revertant colonies. In addition, the mutational spectra were characterized in backgrounds carrying plasmids that encode the umuDC, mucAB, or samAB operon. 4-Nitroquinoline 1-oxide (4-NQO) and furylfuramide (AF-2) induced efficiently -1G, -2(C-G), and +1A frameshift mutations. 4-NQO and AF-2 differed in the ability for the induction of -1A and +1G frameshifts. +1A and -1A frameshift mutations induced by 4-NQO or AF-2 were enhanced by the introduction of the mucAB plasmid, and, to a lesser extent, the umuDC plasmid. The enhancing effect of the umuDC or mucAB plasmid on -1G and -2(C-G) frameshifts was weak or else not observed. 9-Aminoacridine was a potent inducer of +1G, -1G and -1A frameshifts, whereas ICR-191 induced all types of frameshift mutations. A mutation enhancing effect was observed only on ICR-191-induced +1A frameshift mutations by the introduction of the mucAB plasmid. Mitomycin C caused no appreciable induction of frameshift mutations to the tester strains without plasmid. However, all types of frameshifts, except -1G, were induced in the strains carrying the mucAB plasmid. N-methyl-N'-nitro-N-nitrosoguanidine induced all types of frameshift mutations. The mucAB plasmid enhanced mutagenesis in strains designed to detect the addition or loss of A.T base pair, indicating that the formation of +1A and -1A frameshifts was partly dependent on an error-prone SOS repair. Any frameshift mutagenesis was not affected by the samAB plasmid. In general, frameshifts in adenine runs were enhanced more preferentially by the mucAB and umuDC plasmids than frameshifts at runs of guanine were.

Effects of the umuDC, mucAB, and samAB operons on the mutational specificity of chemical mutagenesis in Escherichia coli: II. Base substitution mutagenesis.

Mutational spectra induced by different classes of chemical mutagens including two ultraviolet-mimetic mutagens, an alkylating agent, intercalators, a crosslinking agent, and base analogs were characterized by means of a set of mutant lacZ genes in E. coli. These strains can be used to detect each of two types of transition and four types of transversion, simply by measuring the number of Lac+ revertant colonies. 4-Nitroquinoline 1-oxide induced G.C-->A.T, G.C-->C.G, or G.C-->T.A changes almost equally, whereas furylfuramide and mitomycin C induced only G.C-->A.T transitions and G.C-->T.A transversions, respectively. No base substitutional mutations were detected by the treatment with 9-aminoacridine. A weak stimulation of G.C-->A.T transitions by ICR-191 was observed. Both the G.C-->A.T and A.T-->G.C transitions were induced by N-methyl-N'-nitro-N-nitrosoguanidine and N4-aminocytidine. 5-Azacytidine was a specific inducer of G.C-->C.G transversions. In addition, a comparative study of mutational specificity was performed in the strains bearing either the umuDC, mucAB, or the samAB operon on a multicopy plasmid. Regardless of the kind of mutagen, G.C-->T.A transversions were greatly potentiated by the introduction of plasmids in the order of pGW1700 (mucAB) > pSE117 (umuDC) > or = pYG8011 (samAB). Besides G.C-->T.A transversions, the introduction of pGW1700, but not pSE117 and pYG8011, enhanced the mutations of A.T-->C.G and A.T-->T.A transversions. The mucAB plasmid also enhanced the G.C-->A.T transitions and G.C-->C.G transversions induced by some mutagens.

2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) is a weak in vivo clastogen as revealed by the micronucleus assay.

The in vivo clastogenicity of 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) was examined in the micronucleus test using peripheral blood from three mouse strains (ICR, CD-1, and MS/Ae) and bone marrow from one rat strain (Sprague-Dawley). Doses up to the maximum tolerated were tested. The chemical was given once, twice, thrice, or four times via either the i.p. or p.o. route. Under some conditions, ICR and CD-1 mice showed an increased frequency of micronucleated reticulocytes, but definite conclusions were difficult to draw because the increases were very slight. MS/Ae mice showed a markedly elevated micronucleated reticulocyte frequency after the double and triple ip treatments. Rats showed a slightly but statistically significantly increased frequency of micronucleated polychromatic erythrocytes after double i.p. treatments. These results indicate that AF-2 is a weak in vivo clastogen.

The mutational specificity of 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF2) in the lacI gene of Escherichia coli.

We have determined the mutational specificity of the 5-nitrofuran derivative furylfuramide (AF2) in the lacI gene of Escherichia coli. Treatment of a delta uvrB, pKM101 strain with 1 M AF2 yielded a mutation frequency approximately 300 times greater than that of untreated controls. Of the 165 AF2-induced mutants analysed by DNA sequencing, 145 were base substitution mutations, 11 were frameshifts, and the remainder small deletions, tandem base substitutions and complex mutations. Base substitution occurred primarily (greater than 93%) at G:C base pairs. The proportions of the various mutations are very similar to those that have been reported for AP sites. We suggest that the principal mechanism for AF2 mutagenesis is the formation of an adduct which depurinates to yield AP sites that serve as a substrate for error-prone repair. Seventy-two of the mutations occurred at four 5'-TGC-3' sites. The majority (10/11) of the frameshift mutations occurred at one such hotspot and could have been templated by an inverted repeat less than 100 bp removed from the site of the mutation.

Different effect of hyperthermia and heat shock on the action of quinolone drugs versus some mutagens against chloroplasts of Euglena gracilis.

Hyperthermia (37 degrees C permanently) and heat shock (42 degrees C for 10 min, and then 27 degrees C) retarded the elimination of chloroplasts from the flagellate Euglena gracilis induced by quinolone antibacterial chemotherapeutics (OA, NA, Cnx, Ofx, Cpfx, Enx, Nfx) in comparison with their action at 27 degrees C. In the case of OA, NA, and Cnx those hyperthermic conditions completely blocked their action against chloroplasts. On the other hand, both temperature regimes accelerated the antichloroplast activity of the mutagens/carcinogens nitrosoguanidine and furylfuramide.

Initiating potential of 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and 3,3',4',5,7-pentahydroxyflavone (quercetin) in two-stage mouse skin carcinogenesis.

Tumor initiating activity of 3,3',4',5,7-pentahydroxyflavone (quercetin), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and 2-(2-furyl)-3-(5-nitro-2-furyl) (AF-2) acrylamide) were tested in a two-stage mouse skin carcinogenesis model using 12-O-tetradecanoyl phorbol-13-acetate (TPA) as a promoter. These compounds dissolved in dimethyl sulfoxide were topically applied twice weekly for 5 weeks on the dorsal skin, and then followed by TPA for 47 weeks. The total initiating dose was 100 mg for each compound. 7,12-Dimethylbenz(a)anthracene (DMBA) at a total dose of 100 micrograms was used as a positive control compound. AF-2 induced skin tumors in 35% of the mice (average of 0.4 tumors/mouse), HBA in 15% in (0.2/mouse), BHT in 13% (0.13/mouse) and quercetin in 5% (0.1/mouse). No tumors appeared in the groups treated with either test chemicals alone or TPA alone. Statistical analysis according to either Fisher's exact test or Peto's trend test revealed significant differences for tumor appearance in the AF-2/TPA and BHA/TPA followed by TPA groups as compared to in the DMSO/TPA group. The results indicate that AF-2 and BHA have weak tumor initiating activity on mouse skin, but such effects are not apparent for BHT or quercetin.

Enhancement of toxicity from N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea in V79 spheroids by a nitrofuran.

Hypoxic cell radiosensitizers enhance the cytotoxicity of several common chemotherapeutic agents in vitro and in vivo. Although this process has generally been called sensitization, few studies have documented true potentiation. We have used Chinese hamster V79 spheroids to study chemosensitization and fluorescence-activated cell sorting to specifically evaluate the roles of sensitizer binding and hypoxia in the effect. By using the median effect analysis to quantify the interactions of the agents, we conclude that marked potentiation can indeed be achieved. Somewhat greater potentiation was observed at increased depths within the spheroids, but the relative change was less than predicted on the basis of the decreasing oxygen tension. Further, increased toxicity did not necessarily lead to increased chemopotentiation, nor was potentiation directly related to the metabolism/binding of the nitrofuran. Thus, chemopotentiation is clearly a complicated process, highly dependent upon the sensitizer to antitumor drug ratio and the exposure conditions.

Studies on environmental chemical carcinogenesis in Japan.

The historical background of studies in Japan on chemical carcinogenesis from environmental sources is described from personal experience.

Toxicological significance of hepatic responses to furylfuramide(AF-2) in the rat.

In male rats fed furylfuramide [2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide, AF-2] at a dietary level of 0.1%, a marked increase in the liver weight was observed with a concomitant reduction of microsomal mixed function oxidase activity. To clarify the significance of these hepatic responses to furylfuramide, a series of biochemical parameters that reflect hepatotoxicity was measured. The most significant elevations were seen in hepatic glycogen and glucose 6-phosphate dehydrogenase levels over 29-day feeding interval. A marked decrease was observed in mixed-function oxidases and glucose 6-phosphatase activities. Moderate decreases were seen in several other parameters, especially in the early phase of feeding. Measurement of serum parameters showed significant elevation in cholesterol and a transient increase in serum glutamic pyruvic transaminase. A comparative study demonstrated small differences between furylfuramide and carbon tetrachloride in their effects on hepatic parameters. The furylfuramide-induced changes appear to be reversible within 14 days after withdrawal of furylfuramide from the diet. Reduction of mixed-function oxidase activity by furylfuramide might be caused partly by the elevation of heme degrading enzyme. The results indicate a dysfunction of drug-metabolizing activity and acute and slight toxication of the liver by furylfuramide.

Teratogenic effects of carcinogenic agents on limb regeneration in the Japanese newt Cynops pyrrhogaster.

Normal regeneration of the amputated forelimb of the Japanese newt Cynops pyrrhogaster and regeneration after a single intraperitoneal injection of three potent mutagenic/carcinogenic agents was investigated. Three dose levels of each agent and controls were tested for teratogenicity in this newt model with the following chemicals: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-quinoline-1-oxide, and 2-(2-furyl)-3-(5-nitrofuryl)acrylamide. These chemicals were administered at 10 days (late dedifferentiation stage), 20 days (late bud stage), and 30 days (early digits stage) after amputation at the midforelimb. A total of 628 newts, with 16-20 animals per group, were used. Normal forelimb regeneration in Cynops pyrrhogaster closely paralleled that reported for other species. A variety of deformities, including syndactyly, polydactyly, oligodactyly, brachydactyly, and digital branching, were occasionally observed in control regenerating forelimbs, with syndactyly occurring at highest incidence (17.5%). All three mutagens at all tested dose levels enhanced the incidence of teratogenic changes, though increases were not always statistically significant. MNNG, particularly when administered at the time of initial chondrogenesis (20 days, late bud stage), was especially teratogenic. The type of forelimb deformity was not mutagen-specific in this experiment. As Cynops pyrrhogaster is easily and inexpensively maintained and tolerates surgery well, this model with the regenerating forelimb should prove useful for further studies on teratogen screening. Also, studies directed toward mutagenic and epigenetic effects of exogenous agents on rapidly proliferating and differentiating tissues can be investigated with this model, which obviates transplacental excursion and metabolism of test compounds.

Mutagenic effects of two nitrofuran food preservatives.

Two structurally related food additives, 3-(5-nitro-2-furyl)acrylic acid (5-NFAA) and 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) showed a marked mutagenic effect on Salmonella typhimurium strains TA100 and TA98 and Escherichia coli WP2 uvrA+ and WP2 uvrA. On the molar basis 5-NFAA was about two orders of magnitude less effective than AF-2. In Salmonella typhimurium TA100 anaerobic conditions stimulated the mutagenic effect of 5-NFAA which was more pronounced in nitrofuran-reductase deficient strain of Salmonella typhimurium TA100 FR50. 5-NFAA increased the number of isoleucine revertants and induced mitotic recombination at tryptophan, threonine and adenine loci of the diploid strains Saccharomyces cerevisiae a/b and Saccharomyces cerevisiae SBTD. Activity of 5-NFAA was lower than that of AF-2. The test on sex-linked recessive lethal mutations in Drosophila melanogaster indicated that only 5-NFAA is mutagenic, increasing the mutation frequency about 10-fold above the control. Results with AF-2 fell within the control range.

Carcinogenic, Mutagenic, and Comutagenic Aromatic Amines in Human Foods.

Three recent topics related to possible exposure of humans to mutagenic and carcinogenic aromatic amines and related compounds in foods are reviewed. A food additive, AF-2,2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide, was first demonstrated to be mutagenic in Escherichia coli WP-2 and then proved to be carcinogenic in experimental animals. This is an example of prediction of the carcinogenicity of a compound from results of short-term microbial tests. Pyrolysates of amino acids, proteins, and foods high in a protein contain many heterocyclic aromatic amine compounds. For example, a tryptophan pyrolysate contains two derivatives ofamino-gamma-carboline(pyridoindole), and a glutamicd acid pyrolysate contains two derivatives of djipyridoimidazole. These compounds are strong frameshift mutagens in Salmonella typhimurium. Some of them were carcinogenic in an in vitro transformation test and were also carcinogenic when injected sc into hamsters and rats and when given orally to mice. Carcinogenic aromatic amines, such as aniline, and o-toluidine and yellow OB were demonstrated to be mutagenic in the presence of the beta-carboline, norharman, with S-9 mix. Diphenylnitrosamine was also mutagenic in the presence of norharman, which is present in tobacco tar and broiled food. These mutagenicities of aniline, o-toluidine, yellow OB, and diphenylnitrosamine are discussed in relation to an evaluation of compounds as environmental carcinogens from the results of short-term microbial tests.

Malignant transformation of cryopreserved early passage syrian golden hamster cells by 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide.

2-(2-Furyl)-3-(5-nitro-2-furyl)-acrylamide (AF-2) induced the malignant transformation of secondary cultures of Syrian golden hamster embryo cells prepared from cryopreserved primary cells. Transformed cells grew in semi-solid agar medium and formed sarcomas when inoculated subcutaneously into non-immunosuppressed suckling hamsters.