Screening for the mutagenicity of nitro-group containing hypoxic cell radiosensitizers using Salmonella typhimurium strains TA 100 and TA 98

A series of sixteen 2-, 4- and 5-nitroimidazoles, four nitrobenzenes, five nitrofurans, and a nitropyrrole, most of which have been studied previously as hypoxic cell specific radiosensitizers, have been screened for their mutagenicity using the Salmonella typhimurium strains TA 100 and TA 98 developed by Ames and co-workers. Most of these compounds were mutagenic and had a one to two order of magnitude greater mutagenicity towards TA 100 (base-pair substitution sensitive) than TA 98 (frame-shift sensitive). The spectrum of mutagenic efficiencies for the drugs which was observed could be correlated to some extent with the electron affinity of these compounds. Exceptions to this correlation may indicate drugs of interest for further studies both as mutagens and hypoxic cell radiosensitizers.     

Mutagenicity of potentially carcinogenic mycotoxins produced byFusarium moniliforme

The mutagenic behaviour of two potentially carcinogenic mycotoxins produced byFusarium moniliforme was investigated in theSalmonella mutagenicity test using tester strains TA97a, TA98, TA100, and TA102. The mutagenic response obtained with fusarin C (1, 5, and 10μg/plate) against tester strains TA98 and TA100 in the presence of microsomal activation confirmed previous observations on the mutagenic behaviour of this mutagen while that obtained against TA97a is reported for the first time. No dose-response relationship could be detected for the concentration levels (0.2, 0.5, 1, 5, 10 mg/plate) tested for FB₁, FB₂, and FB₃ against any of the tester strains used in either the plate incorporation and / or the pre-incubation tests. A cytotoxic effect was obtained at concentration levels of 5 and 10mg/plate in the absence of the microsomal activation mixture. From the studies it became evident thatF moniliforme produces two compounds, a mutagenic compound, fusarin C which has been shown to lack carcinogenic activity in rats and the non-mutagenic fumonisin B mycotoxins of which FB₁ is known to be responsible for the hepatocarcinogenicity of the fungus in rats.     

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.     

Mutagenicity of vinyl chloride,chloroethyleneoxide, chloroacetaldehyde and chloroethanol

Exposure of S. typhimurium strains TA 1530, TA 1535 and G-46 to vinyl chloride increased the number of his⁺ rev./plate 16, 12 or 5 times over the spontaneous mutation rate. The mutagenic response for TA 1530 strain was enhanced 7, 4 or 5-fold when fortified S-9 liver fractions from humans, rats or mice were added. In TA 1530 strain, chloroacetic acid showed only toxic effects, while chloroacetaldehyde, chloroethanol and chloroethyleneoxide caused a mutagenic response. The latter compound was shown to be a strong alkylating agent.     

Mutagenic activity of carcinogenic and noncarcinogenic nitrofurans and of urine of rats fed these compounds

The nitrofurans, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT), nitrofurantoin, 5-nitro-2-furoic acid, 5-nitro-2-furamidoxime, 5-nitrofurfurylidene diacetate and the urine of rats fed these compounds, were assayed for mutagenic activity in Salmonella typhimurium strains TA100 and TA100FR1. All the nitrofurans were mutagenic in the order: AF-2 and FANFT > nitrofurantoin > 5-nitro-2-furamidoxime > 5-nitrofurfurylidene diacetate > 5-nitro-2-furoic acid. Strain TA100 was more sensitive than TA100FR1 to the mutagenic influence of these nitrofurans. Only the urine of rats fed AF-2, FANFT and nitrofurantoin had mutagenic activity. Again, TA100 was more sensitive than TA100FR1. The mutagenicity of the urine was not increased by treatment with β-glucuronidase. AF-2, 2-amino-4-(5-nitro-2-furyl)thiazole (deformylated product of FANFT) and nitrofurantoin were excreted in the urine of rats fed these compounds; whereas the other nitrofurans were not excreted.     

Structural specificity of aromatic compounds with special reference to mutagenic activity in Salmonella typhimurium — a series of chloro- or fluoro-nitrobenzene derivatives

The mutagenicity of 21 chloro- or fluoronitrobenzene compounds and 9 chloro- or fluorobenzene compounds in Salmonella typhimurium (strains TA98, TA1538, TA1537, TA100 and TA1535) was examined. The tests were carried out under the conditions of absence and presence of liver microsomal activation.15 nitro-group compounds had mutagenic activity; above all, compounds of fluoronitrobenzene were mutagenic for both types of strain. On the other hand, chloronitrobenzene compounds were mutagenic for base-pair substitution strains only. Mutagenic activity was exhibited by all compounds having a chloro or fluoro substituent at the para and ortho position in the nitrobenzene nucleus. All compounds without a nitro substituent showed no mutagenic activity.     

UKEMS trial compounds: In vitro bacterial mutagenicity

4CMB, 4HMB and BC were examined in the Ames test using Salmonella typhimurium TA1535, TA1537, TA1538, TA98 and TA100. 4CMB was mutagenic for all of the indicator strains, 4HMB was inactive and BC was weakly mutagenic for TA100 only.     

Mutagenicity of the phenacetin metabolites: N-hydroxy-p-phenetidine and nitrosophenetol in S. typhimurium TA100 and derivatives deficient in nitroreductase or O-acetylase: probes for testing intrabacterial metabolic activation

Two mutagenic metabolites of phenacetin, p-nitrosophenetol and N-hydroxy-p-phenetidine, were tested in S. typhimurium strains TA100, its nitroreductase-deficient derivative TA100NR, and O-acetylase-deficient strains TA100 Tn5-1-8-DNP1011 and -DNP1012 in the presence or absence of an exogenous metabolic activation system. The results indicate that bacterial nitroreductase(s) and O-acetylase(s), shown to be involved in the conversion of certain nitroarenes, are not required for the intrabacterial activation of the two phenacetin metabolites to bacterial mutagens. In view of the low reactivity of nitrosoarenes towards nucleophiles at neutrality, the mechanism by which they exert such a high mutagenic effect in S. typhimurium strains remains to be clarified, but is discussed.     

Mutagenicity to Salmonella typhimurium of some Aspergillus and Penicillium mycotoxins

17 mycotoxins produced by various Aspergillus and Penicillium species were screened for their mutagenic activity to Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, both with and without metabolic activation. Austdiol, austocystins A and D, kojic acid and viridicatumtoxin were found to be mutagenic after metabolic activation, while austdiol was also mutagenic per se. Aflatoxin B₁, sterigmatocystin and versicolorin A, which were used as positive controls were also mutagenic. No mutagenic activity was evident in the case of citrinin, cyclopiazonic acid, fumitremorgen B, griseofulvin, luteoskyrin, O-methylsterigmatocystin, mycophenolic acid, ochratoxin A, patulin, penicillic acid, secalonic acid D and TR₂-toxin.A good relationship was found between the mutagenic activity, or lack of it, of most of the mycotoxins with existing data on carcinogenicity. Inadequate information on the carcinogenicity of austdiol, austocystins A and D, kojic acid and viridicatumtoxin precluded correlations with mutagenicity to S. typhimurium. The relationship between chemical structure and mutagenicity of the mycotoxins is discussed.     

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.     

Mutagenicity of some commercially available nitro compounds for Salmonella typhimurium

Benzoyl chloride and 53 commercially available aromatic, heterocyclic and aliphatic nitro compounds were tested for mutagenicity in Salmonella typhimurium TA98 and TA100. 34 of 53 nitro compounds (64%) were mutagenic, 4 in TA100 only, 15 in TA98 only, and 15 in both strains. 13 of the heterocyclic derivatives of pyridine, indole, indazole, quinoline, and benzimidazole were mutagenic. 21 of 34 mutagenic nitro compounds were bactericidal. Nitromethane was the only aliphatic tested and was not mutagenic. Benzoyl chloride, a human carcinogen, was mutagenic for TA98.     

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.     

Mutagenicity of five food additives in Ames/Salmonella/microsome test

The mutagenic activity of five food additives (K₂S₂O₅: potassium metabisulphite, KMB; K₂SO₄: potassium sulphate, KS; Na₂SO₃: sodium sulphite, SS; KNO₃: potassium nitrate, KN; NaNO₃: sodium nitrate, SN) were investigated using histidin auxotrophs TA98 and TA100 strains ofSalmonella typhimurium in the presence or absence of S9 mix. The test substance were investigated for their mutagenic effects at non toxic concentrations of 0.83, 1.66, 3.33 and 5.00 mg/plate with and without S9 mix. All the test substances were not mutagenic on TA98 and TA100 strains ofSalmonella typhimurium in the presence or absence of S9 mix except KS and SN. KS and SN showed a weak mutagenic effect on TA100 strain in the absence of S9 mix.     

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.     

The mutagenic activity of 4-chloromethylbiphenyl (4CMB) and Benzyl Chloride (BC) in the bacterial/microsome assay

The mutagenic activity of 4CMB was investigated in agar layer cultures of Salmonella typhimurium TA1535, TA1537, TA1538, TA98 and TA100, and Escherichia coli WP2 and WP2 uvrA. The mutagenic activity of BC was investigated in the Salmonella strains only. Assays were performed both in the absence and in the presence of S9 microsomal fraction obtained from a liver homogenate from rats pretreated with Aroclor 1254.     

Mutagenicity of 4-aminoantipyrine and 4-nitrosoantipyrine, the C-nitroso derivative of antipyrine

The drug antipyrine and its 4-substituted analogs, 4-aminoantipyrine, 4-dimethylaminoantipyrine (aminopyrine) and 4-nitrosoantipyrine were tested for mutagenicity against the screening array of Salmonella typhimurium tester strains TA100, TA98, TA97, TA102 and TA104. Antipyrine and aminopyrine were nonmutagenic to all 5 tester strains even in the presence of S9. 4-Aminoantipyrine was directly mutagenic to TA97 only and the presence of S9 slightly increased its activity. 4-Nitrosoantipyrine was directly mutagenic to all tester strains used and S9 decreased its activity except with strain TA102. The possible long-term hazards of C-nitroso compounds derived from drugs and dietary constituents are discussed in view of their pluripotent direct genotoxicity.     

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.     

4-chloromethylbiphenyl (4CMB), benzyl chloride (BC) and 4-hydroxymethylbiphenyl (4HMB): Reverse mutation tests with Salmonella typhimurium

In this study 4CMB was shown to be a strong, direct-acting, mutagen for S. typhimurium strains TA1538, TA1537, TA98 and TA100. However, for strain TA1535 the compound was only weakly mutagenic. No conclusive evidence of mutagenic activity was seen in tests with BC or 4HMB.     

Mutagenic and genotoxic effects of theophylline and theobromine in Salmonella assay and in vivo sister chromatid exchanges in bone marrow cells of mice.

The mutagenic and genotoxic effects of two methylxanthines, theophylline (TH) and theobromine (TB), were assessed in the Ames mutagenicity assay (in strains TA97a, TA100, TA102 and TA104) and in vivo sister chromatid exchanges (SCEs) in bone marrow cells of mice. These are the two most commonly used nervous system stimulators throughout the world. TH is used in the long-term treatment of asthma. Bacterial mutagenicity assay showed very weak mutagenic effects of both drugs in Salmonella strains TA102 and TA104 only in certain concentrations when S9 was added to it. No mutagenic effects were observed in any other strains used in this assay either with or without metabolic activation. But results of in vivo SCE assay indicate that these two drugs can induce significant SCE in bone marrow cells of mice.     

Mutagenicity of marijuana and Transkei tobacco smoke condensates in the salmonella/microsome assay

Extracts and smoke condensates of marijuana, Transkei home-grown tobacco and also commercial cigarette tobaccos were assayed for their mutagenic activity to Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538, both with and without metabolic activation. No mutagenic activity was detected in dichloromethane extracts of marijuana and tobacco per se, but all the smoke condensates exhibited mutagenicity with metabolic activation. The only strain not mutated by any of the pyrolyzates was TA1535. Transkei tobacco pyrolyzate proved to be the most mutagenic, followed by marijuana, pipe and cigarette tobacco. Mutagenicity was positively associated with the nitrogen content of the various products. The potent mutagenic action of marijuana smoke condensate, coupled with a condensate yield of more than 50% higher than that of cigarette and pipe tobacco, indicates a high carcinogenic risk associated with marijuana smoking.