Optimum associations of tester strains for maximum detection of mutagenic compounds in the Ames test

The Ames test is now widely used as a short-term test for the detection of mutagens. Different strains are available with various genetic characteristics, and in the past decade various authors have recommended different associations of strains to give maximum detection potential. However, few studies have been done to compare the sensitivity of individual strains towards a wide range of compounds in a single study. In order to define the best association of strains for screening or regulatory purpose, we have tested 103 direct mutagens (reference genotoxins or in-house compounds) on 7 strains of Salmonella typhimurium: TA1535, TA1537, TA1538, TA97, TA98, TA100 and TA102. 126 different associations of strains have been studied in terms of sensitivity and percentage overlap. Optimum associations of 2, 3, 4 or 5 strains included strains both with and without plasmid pKM101. However, the specificity of detection is greatly diminished by the presence of plasmid pKM101 in the strain, as shown by the high degree of overlap in associations constituted entirely of strains containing the plasmid. The association of strains TA1538 and TA100 detected 86% of the chemicals tested and is therefore recommended for large-scale screening. A rate of detection of 100% was obtained when 6 strains were used. The best associations of 4 and 5 strains, which detected 97 and 99% chemicals respectively, all contained strains TA1537, TA1538 and TA102. Finally, the associations of 4 strains (TA1537, TA1538, TA100, TA102) or 5 strains (TA1535, TA1537, TA1538, TA97, TA102) seemed well adapted to the optimum detection of mutagenic compounds.     

Use of recombinant DNA techniques in cloning DNA repair genes and in the study of metagenesis in mammalian cells

18 polycyclic aromatic hydrocarbons (PAHs) and 7 quinones were tested for mutagenicity using Salmonella typhimurium TA97, TA98 and TA100 with or without metabolic activation. In the presence of metabolic activation, TA97 was more susceptible to mutation than either TA98 or TA100 by many of PAHs tested. PAHs such as 1-methylphenanthrene, fluoranthene, pyrene, benzo[a]pyrene, benzo[e]pyrene and perylene had high mutagenic effects on TA97 in the presence of metabolic activation. 1,6- and 1,8-pyrenequinones were also highly mutagenic on TA97 in the presence or absence of metabolic activation. It appears that pyrene is mutagenic through its metabolic conversion to pyrenequinones.     

Mutagenic activity of pyrolysates of cyanocobalamin and some other water-soluble vitamins in the model system with the Salmonella/mammalian microsomes

Pyrolysates of cyanocobalamin, thiamine hydrochloride, riboflavin, pyridoxine hydrochloride, and ascorbic acid were tested for mutagenicity in the histidine-requiring mutants Salmonella typhimurium TA98 and TA100. Each vitamin was sealed in a glass tube and heated at 100-600 degrees C in a muffle furnace. Methanol-chloroform extracts of the pyrolysate of each vitamin tested did not show any mutagenicity in either TA98 or TA100 without rat liver 9000 x g supernatant fraction (S9) added. In the presence of S9, the B-group vitamins (cyanocobalamin, thiamine hydrochloride, riboflavin, and pyridoxine hydrochloride) were all mutagenic in TA98 and TA100, with the highest activity among the vitamins tested found in the pyrolysate of cyanocobalamin. The pyrolysate of 0.25 mumole cyanocobalamin produced 3200 revertants, while the pyrolysates of 0.25 mumole thiamine hydrochloride and riboflavin produced only 910 revertants, and the pyrolysate of pyridoxine hydrochloride did not show any mutagenicity at that amount. The mutagenicity was generally more active to TA98 than to TA100, indicating that frameshift-type mutagens were contained in the pyrolysates. The pyrolysate of ascorbic acid did not show any mutagenic activity in either TA98 or TA100 under the present experimental conditions.     

Enhancement of the mutagenicity of IQ and MeIQ by nitrite in the Salmonella system.

The fried food mutagens IQ, MeIQ, Glu-P-1 and Trp-P-2 were treated with nitrite at pH 3.0 for 1 h at 37 degrees C. The resulting reaction mixtures were tested for mutagenicity towards Salmonella typhimurium TA97, TA98, TA100 and TA1535. Glu-P-1 and Trp-P-2 were readily converted to weak or non-mutagenic deaminated compounds, whereas IQ and MeIQ were converted to extremely strong mutagenic derivatives in both the presence and the absence of rat liver S9 mix. The mutagenicity of MeIQ in TA98 was enhanced by nitrite up to 3-fold, while that of nitrosated MeIQ was further enhanced by S9 mix up to 15-fold. The nitrosation products of MeIQ were resolved into 7 bands by TLC on silica gel plate. Bands I, III, V and VI were highly mutagenic to both TA98 and TA100. The experimental results suggest that the non-enzymatic formation of direct-acting mutagens from indirect-acting mutagens such as IQ or MeIQ might be physiologically important, especially with regard to the etiology of human gastrointestinal tract tumors.     

Absence of mutagenic activity of ticlopidine in the Ames Salmonella/microsome test

Ticlopidine hydrochloride, 5-(o-chlorobenzyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine hydrochloride, a platelet aggregation inhibitor, was tested for mutagenic activity in the Ames Salmonella/mammalian microsome test. Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 were employed. Two of these strains (TA1535 and TA100) are sensitive to base-pair substitution mutagens, and the remaining 3 are sensitive to frame-shift mutagens. There was no evidence that ticlopidine hydrochloride had any mutagenic activity either in the presence or absence of a liver microsomal supplement.     

Influence of the Uvr repair system on the mutagenicity of antiparasitic drugs

One amebicide (chloroquine diphosphate) and 2 anthelmintic compounds (niclosamide and pyrvinium pamoate) were found to be mutagenic for Salmonella typhimurium TA1537, TA1538, TA100 and TA98 Uvr- strains respectively. Drugs tested on homologous Uvr+ strains (TA1977, TA1978, UTH8414 and UTH8413) showed decreased mutagenic activity of the compounds. This indicates that premutational damage induced by the drugs was totally or partially repaired. Furthermore, results obtained in the present study suggest that niclosamide and pyrvinium pamoate induce premutational lesions by adduct formation, and that chloroquine diphosphate, known as an intercalating agent, behaves as an adduct-forming compound as regards its effects on Uvr- and Uvr+ S. typhimurium strains.     

Peroxidative activation of 3,3'-dichlorobenzidine to mutagenic products in the Salmonella typhimurium test

The direct and H2O2-dependent mutagenicity of 3,3'-dichlorobenzidine (DCB) were compared in Salmonella tester strains TA98, TA98/1,8-DNP6, TA100 and TA102 using the Ames test. DCB exhibited both direct and H2O2-dependent mutagenicity to both tester strains TA98 and TA98/1,8-DNP6. This H2O2-dependent mutagenicity of DCB was prevented by horseradish peroxidase. DCB, in contrast to its effects in tester strains TA98, was not mutagenic to TA100 and TA102 either directly or in the presence of H2O2. These results suggest that mechanisms, perhaps enzymes endogenous to tester strains TA98, may play a role in the activation of DCB.     

Chemical defense in ascidians of the didemnidae family

Fluorescent analogues (DB1 and TA1) of the secondary metabolites didemnin B (DB) and tamandarin A (TA) were synthesized to investigate the potential chemical defense mechanisms of tunicates in the family Didemnidae. These compounds were found to alter predator-prey relations. Five species of freshwater fish and one marine fish, the damselfish Amphiprion ocellaris, were acclimated to a diet of mosquito larvae. Fish showed an immediate, negative reaction to mosquito larvae treated with >/=5 ng of DB1 or TA1, with consumption of larvae resulting in regurgitation. Both freshwater and marine fish learned to avoid tainted prey by associating species of larvae with "distaste". Distaste for a given organism also arose when depsipeptides DB1 or TA1 were transferred to the fish from the surrounding medium. Fluorescence microscopy in fish indicated that a similar processing and localization followed ingestion and absorption of DB1 or TA1. Fluorescent labeling of DB or TA provided an ideal tool to conduct short-term studies of predator-prey relationships between fish and marine invertebrate larvae.     

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.     

Mutagenic activity of Fusarium moniliforme isolates in the Salmonella typhimurium assay.

A total of 33 isolates of Fusarium moniliforme from several food or feed crops were grown on sterile cracked corn, and chloroform-isopropanol extracts were assayed for mutagenic activity in the Salmonella typhimurium-microsome system by using tester strain TA98 or TA100 or both. Extracts of 21 (64%) of the isolates assayed against TA100 were mutagenic. Activities of seven of these extracts were increased markedly with incorporation of the liver homogenate (S-9) into the assay. Seven (33%) of the isolates assayed against TA98 were weakly active, with the liver homogenate having little effect on reversion rates.     

Mutagenic activity of Fusarium moniliforme isolates in the Salmonella typhimurium assay.

A total of 33 isolates of Fusarium moniliforme from several food or feed crops were grown on sterile cracked corn, and chloroform-isopropanol extracts were assayed for mutagenic activity in the Salmonella typhimurium-microsome system by using tester strain TA98 or TA100 or both. Extracts of 21 (64%) of the isolates assayed against TA100 were mutagenic. Activities of seven of these extracts were increased markedly with incorporation of the liver homogenate (S-9) into the assay. Seven (33%) of the isolates assayed against TA98 were weakly active, with the liver homogenate having little effect on reversion rates.     

Comparative lectin-binding and agglutination properties of the strain-specific,TA3-St,and the non-strain-specific,TA3-Ha,murine mammary carcinoma ascites sublines. Further studies of receptors in epiglycanin

The complex carbohydrates at the cell surfaces of two TA3, murine mammary carcinoma ascites sublines (the strain-specific, TA3-St subline and the nonstrain-specific, TA3-Ha line) were compared by binding studies with ¹²⁵I-labelled concanavalin A (con A), Ricinis communis agglutinin (RCA), and eel-serum agglutinin (ESA). The TA3-Ha cell bound equal amounts of con A, 1.5-fold more RCA, and 4-fold more ESA than the TA3-St cell. Binding-inhibition studies by these lectins and two others [wheat-germ agglutinin (WGA) and potato lectin (STA)] suggest complementary binding-sites between con A and both RCA and ESA. Quantitative agglutination studies with the five lectins, and inhibition determinations by both neuraminidase-treated and untreated epiglycanin revealed that TA3-St, but not TA3-Ha, cells were agglutinated by con A, and that epiglycanin inhibited this agglutination, as well as the agglutination of rabbit erythrocytes by con A. The presence of a con A receptor on epiglycanin was also suggested by the binding of epiglycanin to con A-Sepharose, and its specific elution with methyl α-d-manno-pyranoside. TA3-St cells were agglutinated at a 10-15-fold lower concentration of either STA or RCA than TA3-Ha cells, but both cells were agglutinated by the same concentration of WGA and ESA. Inhibition by epiglycanin of agglutination of TA3-St cells by either STA or ESA occurred at a concentration lower than that of TA3-Ha cells, but epiglycanin inhibited RCA agglutination of TA3-Ha cells at a concentration     

Isolation and studies of the mutagenic activity in the Ames test of flavonoids naturally occurring in medical herbs

Quercetin, rhamnetin, isorhamnetin, apigenin and luteolin were isolated from medicinal herbs: Erigeron canadensis L., Anthyllis vulneraria L. and Pyrola chloranta L. The mutagenicity of these naturally occurring flavonoids was tested by the Ames method with S. typhimurium strains TA1535, TA1538, TA97, TA98, TA100 and TA102 in the presence and absence of metabolic activation. Of the above flavonoids only quercetin and rhamnetin revealed mutagenic activity in the Ames test. Quercetin induced point mutations in strains TA97, TA98, TA100 and TA102 of S. typhimurium. The presence of S9 rat liver microsome fraction markedly enhanced the mutagenic activity of quercetin in these strains. Rhamnetin appeared to be a much weaker mutagen in the Ames test. The compound induced mutations in strains TA97, TA98 and TA100 of S. typhimurium but only in the presence of metabolic activation.Comparison of the structure of the studied flavonoids with their mutagenic activity indicates that the mutagenicity of flavonoids is dependent on the presence of hydroxyl groups in the 3′ and 4′ positions of the B ring, and that the presence of a free hydroxy or methoxy group in the 7 position of the A ring also probably contributes to the appearance of mutagenic activity of flavonoids in the Ames test. It also appeared that the presence of methoxy groups, particularly in the B ring of the flavonoid molecule, markedly decreases the mutagenic activity of the compound.     

Evaluation of the mutagenic potential of yangambin and of the hydroalcoholic extract of Ocotea duckei by the Ames test

Ocotea duckei Vattimo is a plant popularly known as "louro-de-cheiro" found in the northeast of Brazil. Traditional medicinal uses of this plant are not known, but recent pharmacological studies with the isolated major constituent yangambin have shown various qualities: platelet activating factor (PAF) antagonist, protective effects against cardiovascular collapse and anaphylactic shock, anti-allergic properties, analgesic activity, and depressant effect in the central nervous system. In this work, the Ames test was used to evaluate the mutagenic potential of the hydroalcoholic extract of O. duckei leaves and of yangambin. Using TA97a, TA98, TA100, TA102 and TA1535 strains of Salmonella typhimurium, positive results were obtained only with the hydroalcoholic extract, with or without metabolic activation. Yangambin was not mutagenic, which is of interest due to its pharmacological properties.     

Non-mutagenicity for Salmonella of the chlorinated hydrocarbons aroclor 1254, 1,2,4-trichlorobenzene, mirex and kepone.

A polychlorinated biphenyl mixture, Aroclor 1254, two commercial grade insecticides, mirex and kepone, and a pesticide breakdown product, 1,2,4-trichlorobenzene were evaluated for mutagenicity and hepatic enzyme induction potential in the Salmonella/microsomal assay. None was found to revert strains TA1535, TA1537, TA98 or TA100 when tested with or without metabolic activation. Liver microsomal extracts (S9) from rats induced with 1,2,4-trichlorobenzene were shown to differ from S9 of either control or Aroclor 1254-induced rats in the capacity to activate 2-aminoanthracene mutagenesis.     

Absence of mutagenic activity of benorylate, paracetamol and aspirin in the Salmonella/mammalian microsome test

Benorylate and its two major hydrolysis products, paracetamol and aspirin were examined for mutagenicity in the Salmonella/mammalian microsome screening test. The compounds were tested in 6 strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA100, TA97 and TA98) in the presence and absence of a rat-liver microsome activation system. Benorylate did not show evidence of mutagenic activity in the 6 strains tested with or without metabolic activation at concentrations ranging from 0.006 to 3 mg per plate. Paracetamol and aspirin likewise did not show any evidence of mutagenic activity at concentrations ranging from 0.1 to 50 mg per plate for the former and 0.01 to 50 mg per plate for the latter.     

Mutagenicity of natural naphthoquinones and benzoquinones in the Salmonella/microsome test

The mutagenicities of naturally occurring naphthoquinones and benzoquinones were tested by the pre-incubation method with Salmonella typhimurium strains TA98, TA100 and TA2637, which all contain plasmid pKM101. 6 of the 16 naphthoquinones tested, i.e., plumbagin, naphthazarin, 2-hydroxy-naphthoquinone, vitamin K3 (menadione), juglone and 7-methyljuglone, were mutagenic to strain TA2637 with metabolic activation. Except for juglone and 7-methyl-juglone, these compounds also had slight mutagenic effects on strain TA98 with S9 mix. All the mutagenic naphthoquinones contain one or two hydroxyl and/or methyl substituents. The naphthoquinone mompain, which has four hydroxyl groups, was not mutagenic. Unsubstituted beta-naphthoquinone, naphthoquinones with a prenyl side chain and all bi-naphthoquinone derivatives tested were non-mutagenic. None of the 13 benzoquinones examined was mutagenic to any of the strains used with or without metabolic activation. These results show that natural naphthoquinones are mutagenic when they have only one or two hydroxyl and/or methyl substituents.     

Mutagenicity of the reaction products of dibenzo-p-dioxin with nitrogen oxides.

Dibenzo-p-dioxin (DD) was made to react with various concentrations of nitrogen oxides in the dark. The mutagenicities of the reaction products were tested using Salmonella typhimurium strains TA98, TA100, TA98NR and TA98/1,8-DNP6 in the presence or absence of a mammalian metabolic activation system (S9 mix). DD-NOx (molar ratios 1:3, 1:6 and 1:18) reaction products exhibited mutagenic potency in strains TA98 and TA98/1,8-DNP6 without S9 mix. In a gas chromatography/mass spectrometry study, 2-nitrodibenzo-p-dioxin (NDD) was identified with authentic sample in the mutagenic reaction products. DD-NOx (1:18) reaction products were reduced by sodium hydrogen sulfide and the reduction mixture was analyzed by HPLC. 2,7-Dinitrodibenzo-p-dioxin (DNDD) and 2,8-DNDD were identified as corresponding diamino-DDs in the reduction mixture. 2-NDD, 2,7-DNDD and 2,8-DNDD were also mutagenic in strains TA98 and TA98/1,8-DNP6 without S9 mix and the mutagenicity of DD-NOx reaction products was largely accounted for by the nitro-DDs.     

Three consistent patterns of response to substituted acridines in a variety of bacterial tester strains used for mutagenicity testing

91 substituted acridines were tested for mutagenicity in 1 strain of Escherichia coli (TA78) and 4 strains of Salmonella typhimurium (TA90, TA1537, TA98 and TA100). In general, compounds fall into 3 groups: (i) inactive in all strains, (ii) active in TA78, TA90 and TA1537, or (iii) active in TA98 and often one or more of the other frameshift strains. Compounds of class iii have previously been shown to differ from the others in causing excisible damage to DNA and in showing an enhanced mutagenic response when the plasmid pKM101 is present.     

Absence of mutagenic activity of benorylate,paracetamol and aspirin in the Salmonella//mammalian microsome test

Benorylate and its two major hydroyssis products, paracetamol and aspirin were examined for mutagenicity in the Salmonella/mammalian microsome screening test. The compounds were tested in 6 strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA100, TA97 and TA98) in the presence and absence of a rat-liver microsome activation system. Benorylate did not show evidence of mutagenic activity in the 6 strains tested with or without metabolic activation at concentrations ranging from 0.006 to 3 mg per plate. Paracetamol and aspirin likewise did not show any evidence of mutagenic activity at concentrations ranging from 0.1 to 50 mg per plate for the former and 0.01 to 50 mg per plate for the latter.